Bridging virology with proteomics to define cell immune signaling upon infection

将病毒学与蛋白质组学联系起来，定义感染时的细胞免疫信号

• 批准号: 9328551
• 负责人: Krystal Kar-Yan Lum
• 金额: $ 4.4万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9328551
• 关键词: Acute Promyelocytic Leukemia; Address; Antiviral Agents; Antiviral Therapy; Appearance; Belief; Binding; Biochemistry; Bioinformatics; Biological Assay; Cell Line; Cell Nucleus; Cells; Cellular Immunity; Cytomegalovirus; Cytomegalovirus Infections; Cytoplasm; DNA; DNA Viruses; Deposition; Diffuse; Goals; Hand; Herpesviridae; Herpesviridae Infections; Herpesvirus 1; Human; IRF3 gene; Immune; Immune response; Immune signaling; Immunofluorescence Microscopy; Individual; Infection; Innate Immune Response; Interferons; Laboratories; Mammalian Cell; Mass Spectrum Analysis; Measurement; Mediating; Modeling; Molecular; Molecular Virology; Monitor; Mutate; Mutation Analysis; Natural Immunity; Nuclear; Nuclear Export; Nucleic Acids; Pathogenicity; Pattern; Phosphotransferases; Property; Proteins; Proteomics; Quantitative Reverse Transcriptase PCR; Recruitment Activity; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Signaling Protein; Site; Solvents; TBK1 gene; Testing; Ubiquitin; Viral; Virus Diseases; crosslink; cytokine; ds-DNA; experimental study; human DNA; imaging study; insight; interdisciplinary approach; knock-down; leptomycin B; live cell imaging; live cell microscopy; marenostrin; mutant; novel; overexpression; receptor; scaffold; sensor; small hairpin RNA; stable cell line; transmission process; viral DNA; virology

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.

项目总结/摘要。哺乳动物细胞具有免疫防御， 病原性病毒感染的进展。近年来，Cristea实验室和其他机构发现， 人类细胞可以在受感染细胞的细胞核中区分病毒DNA和人类DNA。这一发现是在 与以前的信念相反，病毒DNA的传感只发生在缺乏的亚细胞区室中， 人DNA（例如，细胞质），并提供了一个重要的新的研究途径，了解细胞 防御核复制DNA病毒。主要的发现是 干扰素诱导蛋白IFI 16作为病毒DNA的核传感器和内源性和先天性免疫应答的关键效应子 对核复制疱疹病毒，包括单纯疱疹病毒-1（HSV-1）和人类 巨细胞病毒（HCMV）。我们在HSV-1和HCMV感染过程中的研究表明，IFI 16结合 病毒双链DNA通过其HIN 200结构域在细胞核中，通过其pyrin结构域寡聚化， 并激活中央细胞质信号传导轴STING-TBK 1-IRF 3，以诱导抗病毒细胞因子。然而，在这方面， IFI 16如何启动和传播来自细胞核的信号仍然是未知的。本提案的目的是 描述了核IFI 16介导的免疫信号传播到该细胞的机制。 疱疹病毒感染后的信号传导轴。我将采用多学科的方法， 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。用HSV-1和HCMV进行这些实验将有助于鉴定一种病毒， 广泛相关和保守的机制，通过IFI 16信号。首先根据我的初步观察 IFI 16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），我将研究实现这种本地化的方法。具体来说，我将进行 免疫亲和分离以评估IFI 16结构基序对介导相互作用的贡献 在IFI 16和PML-NB之间。然后，我将使用定量蛋白质组学方法来确定 感染期间IFI 16的SUMO化状态。突变分析，免疫荧光显微镜， 接下来将使用定量RT-PCR来评估SUMO化、SUMO相互作用基序 （西姆斯）和IFI 16结构特性对IFI 16定位于PML-NB和下游诱导的影响。 抗病毒细胞因子。最后，我将描述IFI 16相互作用的功能，这是免疫系统特有的。 早期感染时的信号。我的初步研究表明，IFI 16与抗病毒IFIT 1/2/3相关， proteins.我将使用活细胞显微镜，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，这个项目将有助于阐明免疫信号传递的分子机制， 这对于理解细胞免疫和开发新的抗病毒疗法至关重要。