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）：关于细胞用于防御外来DNA的机制，以及这些防御如何在感染期间被病毒对策破坏，还有很多东西有待了解。为了解决这一知识缺口，我们的研究宿主防御外来DNA的重点是牛痘，原型痘病毒，作为一种模式病原体。了解痘病毒如何实现细胞质DNA复制将产生新的见解防御机制细胞用来应对所有外来DNA。为了实现这一目标，我们对牛痘病毒DNA复制的研究发现，痘病毒B1激酶对病毒DNA复制是必不可少的，因为需要它来抑制细胞DNA结合蛋白BAF（自整合屏障因子）。如果没有失活，BAF会与病毒DNA结合，并作为一种免疫抑制剂。 防御牛痘DNA复制。BAF的抗病毒活性可能是通过它的能力， 紧凑和聚合的DNA，它结合;我们假设，这种压缩可能有助于宿主防御的多种方式。例如，压实可能通过空间位阻限制DNA对病毒复制蛋白的可接近性，这与异染色质DNA比常染色质更难接近的方式大致相同。对BAF抗痘病毒活性的进一步研究将对其作用机制产生独特的见解。我们的中心假设是，BAF的作用是通过沉默外来DNA通过压实和触发更广泛的炎症反应。在这方面，我们认为BAF启动组装细胞质DNA：蛋白质支架上的内在防御效应和先天信号通路可以收敛。为了验证我们的假设，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。这些研究将有助于深入了解控制高级细胞质形成的机制。 核蛋白复合物目的2）确定BAF如何通过压缩和DNA损伤反应沉默外源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，但被痘病毒逃避，将是这一目标的中心焦点。目的3）确定BAF如何调节外源DNA的先天信号反应。在这个目标中，我们将测试我们的假设，即BAF：DNA复合物是一个支架上的信号蛋白可以集中，从而产生洞察这些途径的激活的分子相互作用的基础。 在整个研究过程中，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，这将是这种病原体的创新应用。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-宿主相互作用的理解，而且将对理解对DNA的免疫反应具有广泛意义。