Mechanisms mediating immune response upon sensing of nuclear viral DNA

 描述(由申请人提供)：哺乳动物免疫系统识别致病DNA的能力，如来自病毒的DNA，对于启动固有和先天免疫反应是必不可少的。这种识别是由被称为DNA传感器的特殊细胞蛋白质完成的，这种蛋白质结合外来DNA并诱导细胞因子的分泌，以提醒邻近细胞并抑制感染的传播。直到最近，对外来DNA的感觉还被认为只发生在通常没有DNA的亚细胞隔间。然而，这种由来已久的信念未能解释细胞是如何检测到具有核复制能力的DNA病毒的。我们实验室最近对第一个确定的核DNA传感器IFI16进行了表征，这有助于牢固地确立核传感的概念。我们证明了IFI16具有感知疱疹病毒的功能，包括重要的病原体人类巨细胞病毒(HCMV)和1型单纯疱疹病毒(HSV-1)(Li等人)。PNAS 2012；Li等人。细胞寄主微生物2013年)。感觉可以发生在细胞核中的发现为免疫研究打开了一个新的方向，这将增加我们对平衡免疫反应如何维持健康系统，以及它们的错误调控如何导致免疫紊乱、癌症以及病毒引起的发病率和死亡率的理解。然而，一个尚未回答的基本问题是免疫信号是如何从细胞核传播的。我们发现，虽然IFI16在HCMV和HSV-1感染的早期阶段仍然是核的，但IFI16介导的抗病毒细胞因子的诱导需要内质网适配蛋白STING-DNA传感通路的枢纽。从细胞核引发免疫反应的要求和免疫信号传播到细胞质的机制仍然难以捉摸。我们的提案将解决这些重要问题。首先，我们将描述IFI16与病毒DNA在细胞核内快速共定位的机制。我们发现IFI16在感染后被招募到早幼粒细胞白血病(PML)核体(NBS)。PML-NBS与抗病毒反应有关，并被证明定位于疱疹病毒复制的起始点。使用多学科方法，我们将检验我们的假设，即PML-NBS作为IFI16集中与病毒DNA结合的相互作用中心，为免疫信号的启动奠定了基础。其次，我们将使用分子生物学、生物化学、光遗传学和活细胞成像来定义启动核免疫信号所需的IFI16特性。我们 假设信号的启动需要IFI16通过其在PML-NBS的比林结构域进行寡聚。第三，我们将确定细胞核衍生的免疫信号是如何传递到细胞质的。我们发现IFI16与干扰素诱导的IFIT蛋白相互作用，这些蛋白在感染时穿梭到细胞核。我们提出机制研究来验证我们的假设，即依赖IFI16的免疫信号是通过这种相互作用传递到STING中枢的。总而言之，我们的结果将通过定义免疫信号传播所需的基本机制来表征新发现的免疫方面--核感知。

 DESCRIPTION (provided by applicant): The ability of the mammalian immune system to recognize pathogenic DNA, such as DNA from viruses, is essential for the onset of intrinsic and innate immune responses. This recognition is accomplished by specialized cellular proteins called DNA sensors, which bind foreign DNA and elicit the secretion of cytokines to alert neighboring cells and inhibit the spread of infection. Until recently, the sensing of foreign DNA was thought to occur only in subcellular compartments normally devoid of DNA. However, this long-standing belief failed to explain how the cell detects nuclear-replicating DNA viruses. Our lab's recent characterization of the first identified nuclear DNA sensor, IFI16, has helped to firmly establish the concept of nuclear sensing. We demonstrated that IFI16 functions to sense herpesviruses, including the important pathogens, human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) (Li et al. PNAS 2012; Li et al. Cell Host Microbe 2013). The discovery that sensing can occur in the nucleus opens a new direction for research in immunity that will increase our understanding of how balanced immune responses work to maintain a healthy system and how their misregulation leads to immune disorders, cancers, and virus-induced morbidity and mortality. However, a fundamental question that has yet to be answered is how the immune signal is propagated from the nucleus. We found that, while IFI16 remains nuclear during the early stages of HCMV and HSV-1 infections, the IFI16-mediated induction of antiviral cytokines requires the endoplasmic reticulum adapter protein STING-a hub for DNA sensing pathways. The requirements for eliciting an immune response from the nucleus and the mechanism of immune signal propagation to the cytoplasm remain elusive. Our proposal will address these important questions. First, we will characterize the mechanism by which IFI16 rapidly co-localizes with viral DNA in the nucleus. We have discovered that IFI16 is recruited to promyelocytic leukemia (PML) nuclear bodies (NBs) following infection. PML-NBs have been implicated in antiviral response and shown to localize to origins of replication of herpesviruses. Using a multidisciplinary approach, we will test our hypothesis that PML-NBs function as interaction centers where IFI16 concentrates its binding of viral DNA, setting the stage for immune signal initiation. Second, we will use molecular biology, biochemistry, optogenetics, and live cell imaging to define the IFI16 properties required for initiating nuclear immune signals. We hypothesize that signal initiation requires IFI16 oligomerization via its pyrin domain at PML-NBs. Third, we will determine how the nucleus-derived immune signal is transmitted to the cytoplasm. We discovered that IFI16 interacts with interferon-inducible IFIT proteins, which shuttle to the nucleus upon infection. We propose mechanistic studies to test our hypothesis that IFI16-dependent immune signaling is relayed by such interactions to the STING hub. Collectively, our results will characterize a newly discovered aspect of immunity, nuclear sensing, by defining the fundamental mechanisms required for immune signal propagation.