Nuclear Sensing of Herpesviral DNA

疱疹病毒 DNA 的核传感

• 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
• 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

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）：微生物成分的先天感知已被充分证明发生在许多细胞位点，包括细胞表面、细胞质和细胞内囊泡，但对核先天信号传导知之甚少。我们定义了一个系统，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 信号传导机制。我们发现，在这些细胞中诱导 IRF-3 信号传导所需的 IFI16 DNA 传感器位于核内，并且其定位并不在细胞核中。 HSV-1 d109 感染后可检测到变化。我们获得了令人兴奋的新结果，表明环状 GMP-AMP 合酶 (cGAS) DNA 传感器位于人类成纤维细胞的核内，并且 cGAS 和 IFI16 均参与这些细胞中 IRF-3 信号传导的激活。这种双传感器途径代表了核 DNA 传感的潜在新途径。此外，我们还获得了令人兴奋的结果，表明 IFI16 对 HSV-1 立即早期 (IE) 基因表达和复制具有独立于干扰素的限制作用。我们假设 IFI16 充当对核 HSV DNA 的 IRF-3 信号传导反应以及对进入细胞核的裸露 HSV DNA 的染色质反应的 DNA 传感器。在本提案中，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，进一步定义与 IFI16 相互作用的蛋白质。 2. 通过研究 IFI16 的 DNA 结合特性、IFI16 和相关蛋白对病毒染色质结构的影响以及 IFI16 DNA 传感器的诱变来确定 IFI16 限制病毒和外源 DNA 的机制，以识别沉默病毒基因组所需的 IFI16 结构域。 3. 通过定义核cGAS在人成纤维细胞HSV DNA核传感中的作用，定义核IFI16和cGAS诱导先天反应的机制；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 是否刺激 cGAS 的酶活性，定义 IFI16 和 cGAS 在先天信号传导中的关系。这些研究将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途径。其次，这些研究将提供有关外源 DNA 传感与先天免疫反应诱导和外源 DNA 沉默之间联系的进一步机制信息。