Bridging virology with proteomics to define cell immune signaling upon infection

项目摘要/摘要。哺乳动物细胞拥有免疫防御系统，可以立即采取行动阻止 致病病毒感染的进展。近年来，克里斯蒂亚实验室和其他实验室发现 人类细胞可以在感染细胞的细胞核中区分病毒DNA和人类DNA。这一发现发表在 与之前的观点不同的是，病毒DNA的感知只发生在缺乏 人类DNA(例如细胞质)，为理解细胞提供了一条重要的新研究途径 防御可复制核的DNA病毒。主要的发现是对 干扰素诱导蛋白IFI16作为病毒DNA的核感受器和内在和先天的重要效应因子 对包括单纯疱疹病毒-1(HSV-1)在内的核复制疱疹病毒和人类 巨细胞病毒(HCMV)。我们在感染HSV-1和HCMV期间的研究表明，IFI16与 病毒通过其HIN200结构域在细胞核中的双链DNA，通过其比林结构域进行寡聚， 并激活中央细胞质信号轴STING-TBK1-IRF3，诱导抗病毒细胞因子。然而， 目前尚不清楚IFI16如何启动和传播来自细胞核的信号。这项提议的目标是 描述核IFI16介导的免疫信号传播到这个区域的机制 疱疹病毒感染的信号轴。我将使用一种融合蛋白质组学的多学科方法 用分子病毒学、活细胞显微镜和生物化学来确定细胞免疫的机制 对疱疹病毒感染的反应。对HSV-1和HCMV进行这些实验将有助于识别 广泛相关和保守的机制，通过它IFI16信号。首先，根据我的初步观察， IFI16在感染后改变其亚核定位与早幼粒细胞白血病小体相关 (PML-NBS)，我将调查实现这种本地化的方法。具体地说，我将进行 免疫亲和分离以评估IFI16结构基序对相互作用的调节作用 在IFI16和PML-NBS之间。然后，我将使用定量蛋白质组学方法来确定 IFI16在感染过程中的苏莫化状态。突变分析，免疫荧光显微镜，以及 接下来将使用定量RT-PCR来评估SUMO化、相扑相互作用基序的贡献 (SIMS)和IFI16的结构特性对IFI16对PML-NBS的定位和下游诱导 抗病毒细胞因子。最后，我将描述免疫所特有的IFI16相互作用的功能 在早期感染时发出信号。我的初步研究表明，IFI16与抗病毒药物IFIT的1/2/3有关 蛋白质。我将使用活细胞显微镜、基因敲除试验和抗病毒细胞因子测量来评估 IFIT蛋白的核质穿梭及其对STING信号传播的贡献。 总之，该项目将有助于阐明免疫信号传递的分子机制， 这对于了解细胞免疫和开发新的抗病毒疗法至关重要。

PROJECT SUMMARY/ABSTRACT. Mammalian cells possess immune defenses that act immediately to hinder the progression of pathogenic viral infection. In recent years, the Cristea laboratory and others have discovered that human cells can distinguish viral DNA from human DNA in the nuclei of infected cells. This finding was in contrast to the previous belief that sensing of viral DNA takes place only in subcellular compartments that lack human DNA (e.g., cytoplasm), and has provided an important new avenue of research for understanding cellular defense against nuclear-replicating DNA viruses. The principal discovery was the characterization of the interferon inducible protein IFI16 as a nuclear sensor of viral DNA and a crucial effector of intrinsic and innate immunity against nuclear-replicating herpesviruses, including herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). Our studies during infection with HSV-1 and HCMV have indicated that IFI16 binds viral double-stranded DNA in the nucleus through its HIN200 domains, oligomerizes through its pyrin domain, and activates the central cytoplasmic signaling axis, STING-TBK1-IRF3, to induce antiviral cytokines. However, how IFI16 initiates and propagates signals from the nucleus remains unknown. The goal of this proposal is to characterize the mechanism by which nuclear IFI16-mediated immune signals are propagated to this signaling axis upon herpesvirus infection. I will use a multidisciplinary approach that integrates proteomics with molecular virology, live cell microscopy, and biochemistry to define the mechanism of cellular immune response to herpesvirus infection. Performing these experiments with both HSV-1 and HCMV will help identify a broadly-relevant and conserved mechanism through which IFI16 signals. First, given my preliminary observation that IFI16 changes its subnuclear localization upon infection to associate with promyelocytic leukemia bodies (PML-NBs), I will investigate the means through which this localization is achieved. Specifically, I will conduct immunoaffinity isolations to assess the contributions of IFI16 structural motifs to mediating the interaction between IFI16 and PML-NBs. I will then use quantitative proteomic approaches to identify changes in the SUMOylation state of IFI16 during infection. Mutational analyses, immunofluorescence microscopy, and quantitative RT-PCR will be used next to assess the contribution of SUMOylation, SUMO interaction motifs (SIMs), and IFI16 structural properties to both the localization of IFI16 to PML-NBs and the downstream induction of antiviral cytokines. Lastly, I will characterize the function of IFI16 interactions that are unique to immune signaling during early infection. My preliminary studies showed that IFI16 associates with the antiviral IFIT 1/2/3 proteins. I will use live cell microscopy, knockdown assays, and antiviral cytokine measurements to assess the nuclear-cytoplasmic shuttling of IFIT proteins and their contribution to the propagation of the signals to STING. Altogether, this project will help elucidate the molecular mechanisms governing immune signal transmission, which is critical for understanding cellular immunity and for developing novel antiviral therapies.