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 Our unpublished data demonstrates that cytoplasmic BAF:DNA complexes also contain DNA repair proteins.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.
DNA-specific intrinsic and innate immune defenses can be triggered by the genomes of both pathogens and the host, and is therefore of broad biomedical importance. The studies in this application will result in a greater understanding of the molecular basis of immune responses to extrachromosomal DNA. The insights gained will allow for future development of diagnostic tools and therapies targeting both infectious and autoimmune diseases.
|Jamin, Augusta; Wicklund, April; Wiebe, Matthew S (2014) Cell- and virus-mediated regulation of the barrier-to-autointegration factor's phosphorylation state controls its DNA binding, dimerization, subcellular localization, and antipoxviral activity. J Virol 88:5342-55|
|Ibrahim, Nouhou; Wicklund, April; Jamin, Augusta et al. (2013) Barrier to autointegration factor (BAF) inhibits vaccinia virus intermediate transcription in the absence of the viral B1 kinase. Virology 444:363-73|