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

描述（由申请人提供）：关于细胞利用防御外源DNA的机制，以及这些防御在感染期间如何被病毒对策破坏，还有很多有待了解的地方。为了解决这一知识缺口，我们的宿主防御外源DNA的研究集中在牛痘病毒上，它是一种典型的痘病毒，作为一种模型病原体。了解痘病毒是如何实现细胞质DNA复制的，将对细胞用来应对所有外来DNA的防御机制产生新的见解。为了实现这一目标，我们对牛痘DNA复制的研究已经导致发现痘病毒B1激酶对病毒DNA复制至关重要，因为它需要灭活细胞DNA结合蛋白BAF（自整合屏障因子）。如果它没有被灭活，BAF就会与病毒DNA结合，并作为一种

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 Our unpublished data demonstrates that cytoplasmic BAF:DNA complexes also contain DNA repair proteins.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.