Nuclear Sensing of Herpesviral DNA

描述（由申请人提供）：微生物组分的先天感应被充分证明发生在许多细胞位点，包括细胞表面、胞质溶胶和胞内囊泡，但对核先天信号传导知之甚少。我们已经定义了一个系统，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信号传导的机制。我们发现，在这些细胞中诱导IRF-3信号所需的IFI 16 DNA传感器是核的，并且其定位不 在HSV-1 d109感染后可检测到变化。我们有令人兴奋的新结果，表明环GMP-AMP合酶（cGAS）DNA传感器在人成纤维细胞中是核，并且cGAS和IFI 16都参与这些细胞中IRF-3信号的激活。这种双传感器通路代表了核DNA传感的潜在新途径。此外，我们有令人兴奋的结果表明，IFI 16对HSV-1立即早期（IE）基因的表达和复制具有限制性作用，这是独立的干扰素。我们假设，IFI 16作为DNA传感器的IRF-3信号反应的细胞核HSV DNA以及染色质化反应的裸HSV DNA进入细胞核。在本建议中，我们的具体目标是：1.通过构建表达标记的IFI 16的细胞系并使用这些细胞系对来自感染细胞的相关蛋白进行免疫沉淀和质谱研究，进一步确定与IFI 16相互作用的蛋白。2.通过研究IFI 16的DNA结合特性、IFI 16和相关蛋白对病毒染色质结构的影响以及IFI 16 DNA传感器的诱变以鉴定沉默病毒基因组所需的IFI 16结构域，来定义IFI 16限制病毒和外源DNA的机制。3.通过定义核cGAS在人成纤维细胞中HSV DNA的核感应中的作用来定义核IFI 16和cGAS诱导先天性应答的机制;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IFI 16是否刺激cGAS的酶活性来定义IFI 16和cGAS在先天性信号传导中的关系。这些研究将深入了解细胞核中涉及IFI 16和cGAS的潜在双重DNA传感机制，从而定义宿主对核DNA病毒感染的先天反应的新途径。其次，这些研究将提供关于外源DNA传感和诱导先天免疫应答以及外源DNA沉默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.