Intracellular Defenses Against Foreign DNA: Insights From Poxvirus-Infected Cells

针对外来 DNA 的细胞内防御：来自痘病毒感染细胞的见解

• 批准号: 8658190
• 负责人: MATTHEW S WIEBE
• 金额: $ 34.03万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8658190
• 关键词: Address; Adenoviruses; Antiviral Agents; Autoimmune Diseases; Autoimmune Process; Bacteria; Binding; Binding Proteins; Cell Nucleus; Cells; Communicable Diseases; Complex; Cytoplasm; DNA; DNA Binding; DNA Damage; DNA Repair; DNA biosynthesis; DNA repair protein; DNA-Binding Proteins; Data; Defense Mechanisms; Development; Diagnostic; Euchromatin; Fluorescence Microscopy; Future; G22P1 gene; Genome; Goals; HIV; Herpesviridae; Histones; Host Defense; Host Defense Mechanism; Immune; Immune response; Infection; Inflammatory Response; Interferon Activation; Knowledge; Learning; Mediating; Methods; Modeling; Modification; Molecular; Nucleoproteins; Pathway interactions; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Poxviridae; Protein Dephosphorylation; Proteins; Publishing; Research; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Protein; Swelling; System; TBK1 gene; Testing; Vaccinia; Vaccinia virus; Vaccinium; Viral; Virus; Work; barrier-to-autointegration factor; base; cell injury; extrachromosomal DNA; innovation; insight; interest; mutant; novel; pathogen; protein complex; repaired; response; scaffold; sensor; tool; uptake; viral DNA

DESCRIPTION (provided by applicant): Much remains to be learned about mechanisms cells utilize to defend against foreign DNA, as well as how these defenses are subverted by viral countermeasures during infection. To address this knowledge gap, our studies of host defenses against foreign DNA focus on vaccinia, the prototypical poxvirus, as a model pathogen. Understanding how poxviruses achieve cytoplasmic DNA replication will yield new insights into the defense mechanisms cells use to respond to all foreign DNA. Toward this goal, our studies of vaccinia DNA replication have led to the discovery that the poxviral B1 kinase is essential for viral DNA replication because it is needed to inactivate the cellular DNA binding protein BAF (barrier to autointegration factor). If it is not inactivated, BAF binds to viral DNA and acts as a defense against vaccinia DNA replication. BAF's antiviral activity likely occurs via its ability to compact and aggregate DNA to which it binds; we postulate that this compaction may contribute to host defense in multiple ways. For example, compaction likely limits the accessibility of the DNA to viral replication proteins via steric hindrance, in much the same way heterochromatic DNA is less accessible than euchromatin. Further examination of BAF's antipoxviral activity will yield unique insights into its mechanism of action. Our central hypothesis is that BAF acts both by silencing foreign DNA through compaction and by triggering a broader inflammatory response as well. In that regard, we posit that BAF initiates the assembly of cytoplasmic DNA:protein scaffolds on which intrinsic defense effectors and innate signaling pathways can converge. To test our hypothesis we propose three aims. AIM 1) Determine how post-translational modifications regulate assembly of BAF:DNA nucleoprotein complexes. These studies will yield insights into mechanisms controlling the formation of higher order cyto- plasmic nucleoprotein complexes. AIM 2) Determine how BAF silences foreign DNA through compaction and a DNA damage response. Our unpublished data demonstrates that cytoplasmic BAF:DNA complexes also contain DNA repair proteins. Understanding how BAF and DNA repair machinery coordinate the silencing of foreign DNA, but are eluded by poxviruses, will be a central focus of this aim. AIM 3) Determine how BAF regulates innate signaling responses to foreign DNA. In this aim, we will test our hypothesis that BAF:DNA complexes are a scaffold on which signaling proteins can concentrate, thus yielding insight into the molecular interactions fundamental for activation of these pathways. Throughout the course of these studies, both wild-type and mutant vaccinia viruses will be used as tools for the further study of BAF and other DNA-specific defenses, which will be an innovative application of this pathogen. At the conclusion of this work, the insights gained will further our understanding not only of poxviral- host interaction, but will be broadly significant to the understanding of immune responses to DNA in general.

描述（由申请人提供）：关于细胞用来防御外来 DNA 的机制，以及这些防御在感染过程中如何被病毒对抗措施破坏，还有很多东西有待了解。为了弥补这一知识差距，我们对宿主对外源 DNA 防御的研究重点关注牛痘，即典型的痘病毒，作为一种模型病原体。了解痘病毒如何实现细胞质 DNA 复制将为细胞用于响应所有外来 DNA 的防御机制提供新的见解。为了实现这一目标，我们对痘苗 DNA 复制的研究发现，痘病毒 B1 激酶对于病毒 DNA 复制至关重要，因为需要它来灭活细胞 DNA 结合蛋白 BAF（自整合因子屏障）。如果没有灭活，BAF 会与病毒 DNA 结合并充当 防御痘苗 DNA 复制。 BAF 的抗病毒活性可能是通过其 压缩并聚集与其结合的DNA；我们假设这种压缩可能以多种方式有助于宿主防御。例如，压缩可能通过空间位阻限制了 DNA 与病毒复制蛋白的接触，就像异染色质 DNA 比常染色质更难接触一样。对 BAF 抗痘病毒活性的进一步检查将对其作用机制产生独特的见解。我们的中心假设是，BAF 的作用是通过压缩来沉默外源 DNA，并引发更广泛的炎症反应。在这方面，我们假设 BAF 启动细胞质 DNA：蛋白质支架的组装，内在防御效应器和先天信号通路可以在其上汇聚。为了检验我们的假设，我们提出了三个目标。目的 1) 确定翻译后修饰如何调节 BAF:DNA 核蛋白复合物的组装。这些研究将深入了解控制高阶细胞质形成的机制。 核蛋白复合物。目标 2) 确定 BAF 如何通过压缩和 DNA 损伤反应来沉默外源 DNA。我们未发表的数据表明细胞质 BAF:DNA 复合物也含有 DNA 修复蛋白。了解 BAF 和 DNA 修复机制如何协调外源 DNA 的沉默，但被痘病毒所逃避，将是这一目标的核心焦点。目标 3) 确定 BAF 如何调节对外来 DNA 的先天信号反应。为此，我们将测试我们的假设，即 BAF:DNA 复合物是信号蛋白可以集中的支架，从而深入了解激活这些途径的基本分子相互作用。 在整个这些研究过程中，野生型和突变型牛痘病毒都将被用作进一步研究 BAF 和其他 DNA 特异性防御​​的工具，这将是该病原体的创新应用。在这项工作结束时，所获得的见解不仅将进一步加深我们对痘病毒与宿主相互作用的理解，而且对理解 DNA 的免疫反应具有广泛的意义。