Mechanisms mediating immune response upon sensing of nuclear viral DNA

为了建立对DNA病毒感染的内在和先天免疫反应，哺乳动物细胞依赖于 识别病毒DNA为外来分子的特殊蛋白质。一旦与病毒DNA结合，这些 传感器诱导细胞因子的分泌，促使邻近细胞激活它们的防御并抑制 感染的传播。近年来，在对过程管理的理解方面取得了重大进展 病毒DNA传感。与之前的教条相反，我们确定哺乳动物细胞可以区分病毒DNA 从感染细胞细胞核中的自身DNA中分离出来。干扰素诱导蛋白IFI16的特性研究 首个已知的病毒DNA核传感器开启了免疫学的新研究方向，开始揭示 关于细胞如何检测核复制病毒，如疱疹病毒。我们进一步发现，人类 病原体、单纯疱疹病毒1型(HSV-1)和人类巨细胞病毒(HCMV)具有免疫逃逸作用 特异性抑制IFI16的机制。有了这些知识，在之前资助的R01期间，我们的目标是 来剖析核IFI16DNA传感的机制。我们回答了有关地点的问题 当IFI16-病毒DNA结合事件发生时，允许IFI16检测DNA的属性，以及 支持IFI16抗病毒反应的功能相互作用。我们的结果揭示了核DNA传感 依赖于核周与亲本病毒DNA的动态开/关传感器关联。我们 证明了病毒DNA上的IFI16寡聚对于核源性免疫信号是必不可少的。我们 进一步证实，当与病毒DNA结合时，IFI16既触发细胞因子的表达，又抑制 病毒基因表达。我们的提案将解决有关核DNA的几个基本问题 从这些发现中觉察到的。在目标1中，我们将定义驱动动态IFI16的机制 与HSV-1 DNA在核周发生寡聚。我们将测试我们的假设，即该属性是 通过快速和可逆液相冷凝事件的生物物理赋予，并描绘了这些 这些事件支配着先天免疫和病毒复制。接下来，我们将描述这两种机制的特征 抗病毒IFI16在其与DNA结合的下游发挥作用。在目标2中，我们将确定IFI16如何诱导 核DNA传感中的先天免疫信号。我们发现，IFI16与该基因相互作用并激活该基因 DNA依赖蛋白激酶(DNA-PK)全酶对疱疹病毒感染的反应。我们将定义 IFI16和DNA损伤反应如何协调以刺激先天免疫和抗病毒非 同源末端连接。在目标3中，我们将阐明ifi16限制病毒基因的机制。 表情。我们将从功能上表征我们展示的IFI16与染色质调节剂的相互作用 作为HSV-1的限制因子。我们将系统地定义将IFI16连接到病毒基因组的途径 异染色化。总而言之，我们的结果将描述生物学新发现的一个方面 将先天免疫与控制DNA损伤修复和基因表达调控的核过程联系起来。

In order to mount intrinsic and innate immune responses to infections by DNA viruses, mammalian cells rely on specialized proteins that recognize the viral DNA as a foreign molecule. Upon binding to viral DNA, these sensors induce cytokine secretion, prompting neighboring cells to activate their defenses and inhibiting the spread of infection. Recent years have seen significant progress in the understanding of processes governing viral DNA sensing. Contrary to prior dogma, we determined that mammalian cells can distinguish viral DNA from self-DNA in the nuclei of infected cells. The characterization of the interferon inducible protein IFI16 as the first known nuclear sensor of viral DNA has opened a new research direction in immunity, starting to shed light on how cells detect nuclear-replicating viruses, such as herpesviruses. We further uncovered that the human pathogens, herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) have immune evasion mechanisms that specifically suppress IFI16. With this knowledge, during the previously funded R01, we aimed to dissect the mechanisms underlying nuclear IFI16 DNA sensing. We addressed questions regarding where and when the IFI16-viral DNA binding event occurs, the properties that allow IFI16 to sense DNA, and the functional interactions that support IFI16 antiviral responses. Our results uncovered that nuclear DNA sensing relies on dynamic on/off sensor associations with parental viral DNA at the nuclear periphery. We demonstrated that IFI16 oligomerization on viral DNA is essential for nucleus-derived immune signaling. We further established that, upon binding to viral DNA, IFI16 triggers both cytokine expression and suppression of viral gene expression. Our proposal will address several fundamental questions regarding nuclear DNA sensing that have emerged from these findings. In Aim 1, we will define what mechanisms drive dynamic IFI16 oligomerization with HSV-1 DNA at the nuclear periphery. We will test our hypothesis that this property is biophysically conferred via rapid and reversible liquid-phase condensation events, and delineate how these events govern innate immunity and viral replication. Next, we will characterize mechanisms underlying the two antiviral IFI16 functions downstream of its binding to DNA. In Aim 2, we will determine how IFI16 induces innate immune signals upon nuclear DNA sensing. We discovered that IFI16 interacts with and activates the DNA dependent protein kinase (DNA-PK) holoenzyme in response to herpesvirus infections. We will define how IFI16 and the DNA damage response coordinate to stimulate innate immunity and antiviral non- homologous end-joining. In Aim 3, we will elucidate the mechanisms underlying IFI16 restriction of virus gene expression. We will functionally characterize the IFI16 interactions with chromatin modulators that we showed to act as HSV-1 restriction factors. We will systematically define the pathways linking IFI16 to viral genome heterochromatinization. Collectively, our results will characterize a newly discovered aspect of biology that links innate immunity to nuclear processes governing DNA damage repair and gene expression regulation.