Nuclear Sensing of Herpesviral DNA

描述(申请人提供)：微生物成分的先天感应被很好地记录在许多细胞部位，包括细胞表面、胞浆和细胞内小泡，但对核先天信号知之甚少。我们建立了一个系统，用于研究感染单纯疱疹病毒(HSV)1的原代人包皮成纤维细胞(HFF)中IRF-3信号的机制。我们发现，在这些细胞中诱导IRF-3信号所需的IFI16 DNA传感器是核的，其定位不是 HSV-1D109感染后可检测到变化。我们有了令人振奋的新结果，表明环状GMP-AMP合成酶(CGAS)DNA传感器在人成纤维细胞中是有核的，cGAS和IFI16都参与了这些细胞中IRF-3信号的激活。这种双传感器途径代表了一种潜在的核DNA传感新途径。此外，我们有令人兴奋的结果表明，IFI16对HSV-1即刻早期(IE)基因的表达和复制具有限制作用，这种作用不依赖于干扰素。我们假设IFI16既是IRF-3对核HSV DNA的信号反应的DNA传感器，也是对裸露的HSV DNA进入核的显色反应的DNA传感器。在这项建议中，我们的具体目标是：1.通过构建表达标记的IFI16的细胞系，并利用这些细胞系进行免疫沉淀和对感染细胞中相关蛋白的质谱研究，进一步确定与IFI16相互作用的蛋白质。2.通过研究IFI16的DNA结合特性，IFI16及其相关蛋白对病毒染色质结构的影响，以及IFI16 DNA传感器的诱变作用，确定IFI16限制病毒和外源DNA的机制，以确定病毒基因组沉默所需的IFI16结构域。3.通过定义核内cGAS在人成纤维细胞中对HSV DNA的核敏感作用，以及通过确定IFI16和cGAS是否影响彼此的核定位、它们与病毒DNA的结合和/或IFI16是否刺激cGAS的酶活性，来确定核IFI16和cGAS诱导先天性反应的机制。这些研究将为深入了解细胞核中涉及IFI16和cGAS的潜在双重DNA传感机制，从而为宿主对核DNA病毒感染的先天反应定义一条新的途径。其次，这些研究将提供关于外来DNA感知和诱导先天性免疫反应以及沉默外来DNA之间联系的进一步机制信息。

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.