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

描述(由申请人提供)：关于细胞用来防御外来DNA的机制，以及这些防御机制如何在感染期间被病毒对策颠覆，仍有许多有待了解。为了解决这一知识差距，我们对宿主对外来DNA的防御研究集中在牛痘病毒这一典型的典型病原体上，作为一种模式病原体。了解痘病毒是如何实现细胞质DNA复制的，将为细胞用来应对所有外来DNA的防御机制提供新的见解。为了实现这一目标，我们对牛痘病毒DNA复制的研究发现，痘病毒B1激酶是病毒DNA复制所必需的，因为它是灭活细胞DNA结合蛋白BAF(自整合因子屏障)所必需的。如果它没有被灭活，BAF就会与病毒DNA结合并发挥作用 对牛痘DNA复制的防御。BAF的抗病毒活性可能是通过它的能力来实现的 紧凑和聚集它结合的DNA；我们假设这种紧凑可能以多种方式有助于宿主防御。例如，紧凑作用可能通过空间位阻限制DNA对病毒复制蛋白的可及性，就像异染色质DNA比常染色质更难获得一样。进一步研究BAF的抗痘病毒活性将对其作用机制产生独特的见解。我们的中心假设是，BAF既通过致密作用沉默外来DNA，也通过引发更广泛的炎症反应来发挥作用。在这方面，我们假设BAF启动了细胞质DNA的组装：内在防御效应器和天然信号通路可以聚集在其上的蛋白质支架。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核蛋白复合体的组装。这些研究将对控制高阶细胞质形成的机制产生深刻的影响。 核蛋白复合体。目的2)确定BAF是如何通过压实和DNA损伤反应沉默外源DNA的。我们未发表的数据表明，细胞质BAF：DNA复合体也含有DNA修复蛋白。了解BAF和DNA修复机制如何协调外来DNA的沉默，但却被痘病毒避开，将是这一目标的中心焦点。目的3)确定BAF如何调节对外源DNA的先天信号反应。在这个目标中，我们将检验我们的假设，即BAF：DNA复合体是信号蛋白可以集中的支架，从而深入了解这些通路激活的分子相互作用基础。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的意义。

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.