Nuclear Sensing of Herpesviral DNA

疱疹病毒 DNA 的核传感

基本信息

批准号：
9250081
负责人：
DAVID M. KNIPE
金额：
$ 44.35万
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 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9250081
关键词：
Acute Affect Binding Candidate Disease Gene Capsid Cell Line Cell Nucleus Cell surface Cells Chromatin Structure Cyclic GMP Cytosol DNA DNA Binding DNA Virus Infections Dinucleoside Phosphates Disease Fibroblasts Gene Expression Herpesvirus 1 Human Immediate-Early Genes Immune Evasion Immune response Immunoprecipitation Income Infection Inflammatory Innate Immune Response Interferons Link Mass Spectrum Analysis Microbe Mouth Diseases Mutagenesis Nuclear Oral Oral mucous membrane structure Pathway interactions Periodicity Plasmids Process Property Proteins Regulation Repression Resistance Role Signal Induction Signal Pathway Signal Transduction Simplexvirus Site System Vesicle Viral Viral Genes Viral Genome Viral Vaccines Virus chromatin modification gene product improved insight keratinocyte knock-down microbial novel therapeutic intervention novel therapeutics penis foreskin protein complex public health relevance response sensor viral DNA

项目摘要

DESCRIPTION (provided by applicant): Innate sensing of microbial components is well documented to occur at many cellular sites, including the cell surface, cytosol, and intracellular vesicles, but less is known about nuclear innate signaling. We have defined a system for studying the mechanisms of IRF-3 signaling in primary human foreskin fibroblast (HFF) cells infected with herpes simplex virus (HSV) 1. We found that the IFI16 DNA sensor, which is required for induction of IRF-3 signaling in these cells, is nuclear, and its localization does not change detectably upon HSV-1 d109 infection. We have exciting new results showing that the cyclic GMP-AMP synthase (cGAS) DNA sensor is nuclear in human fibroblasts and that both cGAS and IFI16 are involved in activation of IRF-3 signaling in these cells. This dual sensor pathway represents a potential new pathway for nuclear DNA sensing. In addition, we have exciting results showing that IFI16 has a restrictive role on HSV-1 immediate-early (IE) gene expression and replication that is independent of interferons. We hypothesize that IFI16 serves as the DNA sensor for both the IRF-3 signaling response to nuclear HSV DNA as well as the chromatinization response to naked HSV DNA entering the nucleus. In this proposal our specific aims are to: 1. Further define the proteins interacting with IFI16 by construction of cell lines tht express tagged IFI16 and the use of these cell lines for immunoprecipitation and mass spectrometry studies of associated proteins from the infected cells. 2. Define the mechanisms of IFI16 restriction of viral and foreign DNA by studies of the DNA-binding properties of IFI16, the effect of IFI16 and associated proteins on viral chromatin structure, and mutagenesis of the IFI16 DNA sensor to identify the domains of IFI16 needed for silencing of the viral genome. 3. Define the mechanisms of nuclear IFI16 and cGAS induction of innate responses by definition of role of nuclear cGAS in nuclear sensing of HSV DNA in human fibroblasts; and definition of the relationship between IFI16 and cGAS in innate signaling by determining if the proteins affect the nuclear localization of each other, their binding to viral DNA, and/or if IFI16 stimulates the enzymatic activity of cGAS. These studies will provide insight into a potential dual DNA sensing mechanism involving IFI16 and cGAS in the cell nucleus, thereby defining a new pathway for the host innate response to nuclear DNA virus infection. Second, the studies will provide further mechanistic information about the link between foreign DNA sensing and induction of innate immune responses and silencing of the foreign DNA.

描述（由申请人提供）：在许多细胞部位，包括细胞表面，细胞质和细胞内囊泡，对微生物成分的先天感应有充分的文献记录，但对核先天信号传导的了解较少。我们已经定义了一个用于研究原代人包皮成纤维细胞（HFF）细胞中IRF-3信号传导机制的系统在HSV-1 D109感染后可检测到更改。我们有令人兴奋的新结果表明，环状GMP-AMP合酶（CGAS）DNA传感器在人成纤维细胞中是核的，并且CGA和IFI16都参与这些细胞中IRF-3信号的激活。该双传感器途径代表了核DNA传感的潜在新途径。此外，我们还具有令人兴奋的结果，表明IFI16在HSV-1立即（即）基因表达和复制中具有限制性作用，而与干扰素无关。我们假设IFI16是IRF-3信号传导对核HSV DNA的DNA传感器，以及对进入核的裸HSV DNA的染色质反应。在此提案中，我们的具体目的是：1。进一步定义了通过构造细胞系的蛋白质与IFI16相互作用的蛋白质，并使用这些细胞系的使用，并使用这些细胞系进行免疫沉淀和质谱研究的质谱研究。 2。通过研究IFI16的DNA结合特性，IFI16的限制和外源DNA的机制，IFI16和相关蛋白对病毒染色质结构的影响以及IFI16 DNA传感器的诱变，以识别IFI16的域，以识别IFI16的识别。 3。定义核CGA在人类成纤维细胞中HSV DNA核传感中的作用定义核IFI16和CGA诱导先天反应的机制；以及通过确定蛋白质是否影响彼此的核定位，它们与病毒DNA的结合和/或IFI16刺激CGA的酶活性，对先天信号传导中IFI16和CGA之间关系的定义。这些研究将洞悉涉及细胞核中IFI16和CGA的潜在双DNA感应机制，从而定义了宿主对核DNA病毒感染的先天反应的新途径。其次，研究将提供有关外国DNA感应与先天免疫反应的诱导与外源DNA沉默之间联系的进一步机械信息。