BAF: an Intrinsic Host Defense Responsive to Foreign DNA

描述(由申请人提供)：将病原体的遗传物质识别为“外来”会提醒细胞注意入侵者的存在。与此同时，对宿主核酸的不受控制的免疫反应错误地以健康细胞为目标进行破坏，导致自身免疫性疾病。因此，了解我们的免疫系统如何检测外来DNA，以及在没有真正威胁的情况下限制它们的控制机制是至关重要的。在这一应用中，我们证明了自整合障碍因子(BAF)是一种新的DMA特异的宿主防御蛋白。我们已经确定BAF是一种抑制痘病毒DNA复制的因子，并假设BAF对任何细胞质DNA都有反应。正如我们的三个具体目标所概述的那样，我们的主要目标是研究BAF如何促进细胞对外来DNA的识别和反应。在第一个目标中，我们将澄清我们对BAF抑制痘病毒生命周期各个阶段的机制的理解(S)。BAF的突变形式，过度表达或已经耗尽BAF的已建立的细胞系，以及野生型牛痘病毒和温度敏感型病毒都将被利用。第二个目的建立在我们最近的观察基础上，即BAF在转染质粒DNA时重新定位到离散的靶点，并延缓由这些质粒编码的基因的表达，表明BAF针对多个DNA来源。我们将使用一系列方法，包括免疫荧光分析、BAF：质粒共沉淀研究和报告基因分析来表征BAF在外源DNA上的募集及其对结合的抑制作用。第三个目标将集中在两个一般主题上：[1]了解BAF作为DNA传感器的功能是如何通过动态磷酸化来调节的，以及[2]确定BAF对外来DNA的抑制是否伴随着细胞信号通路的激活或与之交叉。总之，本文概述的研究将进一步加深我们对BAF作为一种新的宿主防御蛋白的理解。了解和利用天花和猴痘感染宿主/病原体的相互作用具有重要的生物医学意义。天花是天花的病原体，也是潜在的生物恐怖威胁。探索BAF对外来DNA的反应也将阐明在其他类型的感染或自身免疫性疾病发展过程中发生的事件，并将与建立有效的基因治疗和DNA疫苗策略有关。

DESCRIPTION (provided by applicant): Recognition of a pathogen's genetic material as "foreign" alerts the cell to the presence of an invader. At the same time, an unchecked immune reaction to host nucleic acid incorrectly targets healthy cells for destruction, leading to autoimmune disease. It is therefore critical to understand how our immune systems detect foreign DNA, as well as the control mechanisms that restrain them in the absence of a true threat. In this application, we demonstrate that the barrier to autointegration factor (BAF) is a novel DMA-specific host defense protein. We have identified BAF as an inhibitor of poxviral DNA replication and hypothesize that BAF responds to any cytoplasmic DNA. As outlined in our three Specific Aims, our primary goal is to examine how BAF contributes to the cellular recognition and response to foreign DNA. In the first aim, we will clarify our understanding of the mechanism(s) through which BAF can inhibit various stages of the poxviral lifecycle. Mutant forms of BAF, established cell lines that overexpress or have been depleted of BAF, and both wild-type vaccinia and temperature-sensitive viruses will be utilized. The second aim builds upon our recent observation that BAF relocalizes to discrete foci upon transfection of plasmid DNA, and retards expression of genes encoded by these plasmids, indicating that BAF targets multiple sources of DNA. We will employ a battery of approaches including immunofluorescence analysis, BAF:plasmid co- precipitation studies, and reporter gene assays to characterize the recruitment of BAF to foreign DNA and its inhibitory impact upon binding. The third aim will focus on two general themes: [1] understanding how BAF's ability to function as a DNA sensor is regulated by dynamic phosphorylation, and [2] determining whether BAF's repression of foreign DNA is accompanied by activation of, or intersection with, cellular signaling pathways. In sum, the studies outlined here will further our understanding of BAF as a novel host defense protein. Understanding and exploiting the host/pathogen interactions that accompany infection with variola, the etiological agent of smallpox and a potential bioterrorist threat, and monkeypox, an emerging pathogen, is of significant biomedical importance. Exploring BAF's response against foreign DNA will also illuminate events that occur during other types of infections or the development of autoimmune disease, and will be of relevance to the establishment of effective strategies for gene therapy and DNA vaccination.