Mechanisms mediating immune response upon sensing of nuclear viral DNA

为了启动对DNA病毒感染的内在和先天免疫应答，哺乳动物细胞依赖于 识别病毒DNA作为外来分子的特殊蛋白质。一旦与病毒DNA结合， 传感器诱导细胞因子分泌，促使邻近细胞激活其防御，并抑制细胞因子分泌。 感染的传播。近年来，在理解控制过程方面取得了重大进展， 病毒DNA传感与先前的教条相反，我们确定哺乳动物细胞可以区分病毒DNA， 来自受感染细胞核中的自身DNA。干扰素诱导蛋白IFI 16作为干扰素诱导蛋白的特性研究 第一个已知的病毒DNA核传感器开辟了一个新的免疫研究方向，开始揭示 细胞如何检测核复制病毒，如疱疹病毒。我们进一步发现， 单纯疱疹病毒1型（HSV-1）和人巨细胞病毒（HCMV）等病原体具有免疫逃逸 抑制IFI 16的机制。有了这些知识，在以前资助的R 01期间，我们的目标是 以剖析核IFI 16 DNA传感的机制。我们讨论了关于 以及当IFI 16-病毒DNA结合事件发生时，允许IFI 16感测DNA的性质，以及 支持IFI 16抗病毒反应的功能相互作用。我们的研究结果揭示了核DNA传感 依赖于在细胞核外围与亲本病毒DNA的动态开/关传感器关联。我们 证明了病毒DNA上的IFI 16寡聚化对于核衍生的免疫信号传导是必需的。我们 进一步证实，IFI 16与病毒DNA结合后，会触发细胞因子表达和细胞因子抑制。 病毒基因表达我们的建议将解决几个关于核DNA的基本问题 从这些发现中产生的感觉。在目标1中，我们将定义驱动动态IFI 16的机制 在细胞核周围与HSV-1 DNA的寡聚化。我们将测试我们的假设，即该属性是 通过快速和可逆的液相冷凝事件赋予的生物活性，并描述这些 事件控制先天免疫和病毒复制。接下来，我们将描述这两种机制的特点 抗病毒IFI 16在其与DNA结合的下游起作用。在目标2中，我们将确定IFI 16如何诱导 先天免疫信号对核DNA传感。我们发现，IFI 16可以与 DNA依赖性蛋白激酶（DNA-PK）全酶对疱疹病毒感染的反应。我们将定义 IFI 16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。目的3：阐明病毒基因IFI 16限制性酶切的机制 表情我们将在功能上表征IFI 16与染色质调节剂的相互作用， 作为HSV-1限制因子。我们将系统地确定连接IFI 16和病毒基因组的途径 异染色质化总的来说，我们的研究结果将表征生物学的一个新发现的方面， 将先天免疫与控制DNA损伤修复和基因表达调节的核过程联系起来。

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.