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病原性DNA（如病毒DNA）的能力对于内在和先天免疫应答的发生至关重要。这种识别是由称为DNA传感器的专门细胞蛋白质完成的，它们结合外源DNA并引发细胞因子的分泌，以警告邻近细胞并抑制感染的传播。直到最近，外来DNA的感应被认为只发生在通常缺乏DNA的亚细胞区室中。然而，这种长期存在的信念未能解释细胞如何检测核复制DNA病毒。我们实验室最近对第一个确定的核DNA传感器IFI 16的表征有助于牢固地建立核传感的概念。我们证明了IFI 16具有感测疱疹病毒的功能，包括重要的病原体，人巨细胞病毒（HCMV）和单纯疱疹病毒1型（HSV-1）（Li et al. PNAS 2012; Li et al. Cell Host Microbe 2013）。传感可以发生在细胞核中的发现为免疫研究开辟了一个新的方向，这将增加我们对平衡的免疫反应如何维持健康系统以及它们的失调如何导致免疫紊乱，癌症和病毒诱导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如何从细胞核传播。我们发现，虽然IFI 16在HCMV和HSV-1感染的早期阶段保持核，但IFI 16介导的抗病毒细胞因子的诱导需要内质网适配器蛋白STING-DNA传感途径的枢纽。从细胞核引发免疫应答的要求和免疫信号传播到细胞质的机制仍然难以捉摸。我们的建议将解决这些重要问题。首先，我们将描述IFI 16在细胞核中与病毒DNA快速共定位的机制。我们已经发现IFI 16在感染后被募集到早幼粒细胞白血病（PML）核体（NB）。PML-NB已涉及抗病毒反应，并显示定位于疱疹病毒的复制起点。使用多学科的方法，我们将测试我们的假设，即PML-NBs作为相互作用中心，IFI 16集中其病毒DNA的结合，为免疫信号启动设置阶段。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IFI 16特性。我们 假设信号起始需要IFI 16通过其在PML-NB处Pyrin结构域寡聚化。第三，我们将确定核源性免疫信号如何传递到细胞质。我们发现IFI 16与干扰素诱导的IFIT蛋白相互作用，后者在感染后穿梭于细胞核。我们提出了机制研究来验证我们的假设，即IFI 16依赖性免疫信号通过这种相互作用传递到STING中心。总的来说，我们的研究结果将通过定义免疫信号传播所需的基本机制来表征免疫的一个新发现的方面，即核传感。

 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.