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Nuclear Sensing of Herpesviral DNA

疱疹病毒 DNA 的核传感

基本信息

批准号：
10207393
负责人：
DAVID M. KNIPE
金额：
$ 48.38万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-15 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10207393
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 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 epigenetic silencing 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

项目摘要

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.

项目概要这项研究的长期目标是确定外源病毒或质粒 DNA 的机制。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 与组蛋白无关，但宿主细胞会迅速添加进入细胞核后，异染色质与 HSV 基因组结合。感知细胞因子外源病毒 DNA 的表征很差。我们之前已经证明宿主 IFI16 蛋白可以感知 HSV DNA 并刺激先天反应，在之前的资助期间，我们表明 IFI16 促进 ICP0-null HSV 的表观遗传沉默。 IFI16 是唯一已知可增加 HSV 染色质上的异染色质标记。 ND10 蛋白 PML、Sp100、Daxx 和 ATRX 均具有据报道限制 ICP0 无效突变病毒的复制和基因表达，但没有信息了解它们如何影响 HSV 染色质。我们设计了一种新颖的定量成像分析检测输入病毒 DNA 和宿主因子的工具，我们有令人兴奋的新结果表明感染后 30 分钟 (pi) 时 IFI16 与病毒输入 DNA 以及 ATRX 与病毒的顺序关联注射后 0.5-2 小时输入 DNA。 IFI16 和 ATRX 与输入病毒 DNA 的关联似乎是独立的，并且它们对病毒复制的影响是累加的。因为我们已经证明病毒基因组在注射后 1-2 小时内加载异染色质，IFI16 和/或 ATRX 是强有力的候选者在病毒 DNA 的初始传感和异染色质负载中的作用。我们还用了一个小说小分子筛选以确定抑制 ICP0 特异性反式激活的分子，这将提供重要的 ICP0 功能的探头。在此应用中，我们的具体目标是 1. 确定 IFI16 在早期 HSV DNA 传感和染色质化。 2.确定涉及ATRX的作用和机制和其他 ND10 蛋白参与早期 HSV DNA 染色质化。 3. 定义功能机制通过确定 ICP0 促进降解的机制，ICP0 对宿主 DNA 传感器的作用 IFI16及小分子抑制ICP0依赖性基因作用机制的研究表达。这些研究将为外源 DNA 传感机制提供重要的新基础知识并为疱疹病毒提供新的治疗方法，并利用病毒改进基因传递机制和基于 DNA 的技术。

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（1）

CRISPR-Cas9 Expressed in Stably Transduced Cell Lines Promotes Recombination and Selects for Herpes Simplex Virus Recombinants.

DOI：
10.1016/j.crviro.2022.100023
发表时间：
2022-06
期刊：
Current research in virological science
影响因子：
0
作者：
H. Oh;F. Diaz;Changhong Zhou;Nicholas Carpenter;D. Knipe
通讯作者：
H. Oh;F. Diaz;Changhong Zhou;Nicholas Carpenter;D. Knipe

Cellular sensing of viral DNA and viral evasion mechanisms.

DOI：
10.1146/annurev-micro-091313-103409
发表时间：
2014
期刊：
Annual review of microbiology
影响因子：
10.5
作者：
Orzalli MH;Knipe DM
通讯作者：
Knipe DM

Herpes simplex virus infected cell protein 8 is required for viral inhibition of the cGAS pathway.

DOI：
10.1016/j.virol.2023.05.002
发表时间：
2023-05
期刊：
Virology
影响因子：
3.7
作者：
N. Broekema;Max E. Mertens;Magdalena Angelova;M. H. Orzalli;H. Oh;D. Knipe
通讯作者：
N. Broekema;Max E. Mertens;Magdalena Angelova;M. H. Orzalli;H. Oh;D. Knipe

Nuclear sensing of viral DNA, epigenetic regulation of herpes simplex virus infection, and innate immunity.