BAF: an Intrinsic Host Defense Responsive to Foreign DNA

BAF：对外来 DNA 做出反应的内在宿主防御

• 批准号: 7573755
• 负责人: MATTHEW S WIEBE
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-04 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7573755
• 关键词: Autoimmune Diseases; Binding; Biological Assay; Biosensor; Cell Nucleus; Cells; Cellular biology; Co-Immunoprecipitations; Cytoplasm; DNA; DNA Binding; DNA biosynthesis; DNA-Binding Proteins; Data; Deoxyribonucleases; Development; Environment; Eukaryotic Cell; Event; Funding; Gene Expression; Genes; Genetic Materials; Genetic Transcription; Genome; Goals; Host Defense; Immune; Immune response; Immune system; Immunofluorescence Immunologic; Infection; Invaded; Mass Spectrum Analysis; Mediating; Methods; Molecular; Monkeypox; Mutation; Nuclear Envelope; Nuclear Import; Nucleic Acids; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plasmids; Poxviridae; Precipitation; Property; Protein Kinase; Proteins; Reaction; Recruitment Activity; Regulation; Reporter Genes; Repression; Role; Signal Pathway; Signal Transduction; Smallpox; Source; Specificity; Staging; Sum; TLR9 gene; Temperature; Time; Transfection; Vaccination; Vaccinia; Virus; barrier-to-autointegration factor; base; established cell line; gene therapy; inhibitor/antagonist; mutant; novel; overexpression; pathogen; plasmid DNA; response; sensor; viral DNA

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.

描述（由申请人提供）：将病原体的遗传物质识别为“外来”，使细胞警惕入侵者的存在。与此同时，对宿主核酸的未经检查的免疫反应错误地靶向健康细胞进行破坏，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抑制它们的控制机制至关重要。在本申请中，我们证明了自整合屏障因子（BAF）是一种新的DMA特异性宿主防御蛋白。我们已经确定BAF作为痘病毒DNA复制的抑制剂，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如何有助于细胞对外源DNA的识别和反应。在第一个目标中，我们将阐明我们对BAF抑制痘病毒生命周期各个阶段的机制的理解。将使用突变形式的BAF、过表达或已耗尽BAF的已建立细胞系以及野生型牛痘病毒和温度敏感性病毒。第二个目标建立在我们最近的观察，即BAF重新定位到离散的焦点转染质粒DNA后，并延迟这些质粒编码的基因的表达，表明BAF靶向多个来源的DNA。我们将采用一系列方法，包括免疫荧光分析、BAF：质粒共沉淀研究和报告基因测定，以表征BAF对外源DNA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：[1]了解BAF作为DNA传感器的能力如何通过动态磷酸化调节，[2]确定BAF对外源DNA的抑制是否伴随着细胞信号通路的激活或交叉。总之，这里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。理解和利用宿主/病原体的相互作用，伴随感染天花，天花的病原体和潜在的生物恐怖主义威胁，猴痘，一种新兴的病原体，是显着的生物医学重要性。探索BAF对外源DNA的反应也将阐明在其他类型的感染或自身免疫性疾病的发展过程中发生的事件，并将与建立基因治疗和DNA疫苗接种的有效策略有关。