Forward genetic analysis of Mammalian Resistance to Viral Infection

哺乳动物对病毒感染抵抗力的正向遗传分析

• 批准号: 8678818
• 负责人: BRUCE A BEUTLER
• 金额: $ 42.6万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8678818
• 关键词: Adaptor Signaling Protein; Affect; Africa; Alleles; Blindness; Bunyavirus Infections; CCR5 gene; Categories; Cell physiology; Cells; Collaborations; Complex; Computer Simulation; Congenital Abnormality; Cytomegalovirus; Cytomegalovirus Infections; Dendritic Cells; Disease; Economics; Embryo; Endosomes; Fibroblasts; Funding; Genes; Genetic; Genomic DNA; Goals; Grant; Gray unit of radiation dose; HIV; Health; Herpesviridae Infections; Homologous Protein; Host Defense Mechanism; Host resistance; Human; Immune response; Induced Mutation; Infection; Institutes; Instruction; Interferon Type I; Interferons; Life; Life Cycle Stages; Mammalian Cell; Maps; Massive Parallel Sequencing; Membrane Protein Traffic; Methods; Modeling; Molecular; Molecular Target; Murid herpesvirus 1; Mus; Mutagenesis; Mutation; Natural regeneration; Nucleic Acids; Pathway interactions; Phenotype; Pluripotent Stem Cells; Population; Predisposition; Primates; Proliferating; Protein Isoforms; Proteins; Resistance; Resolution; Rift Valley fever virus; Role; Signal Transduction; Systemic Lupus Erythematosus; TLR7 gene; Technology; Therapeutic Intervention; Time; Variant; Viral; Viral Pathogenesis; Virus; Virus Diseases; Work; base; cell motility; cell type; combat; cost; design; empowered; exome; gene induction; genetic analysis; human disease; interest; nervous system disorder; new technology; pathogen; prevent; protein protein interaction; resistance allele; resistance factors; response; screening; segregation; simulation; stem cell technology

We will extend our successful forward genetic analysis of susceptibility to mouse cytomegalovirus (MCMV) nfection, and institute a new screen for cell-autonomous resistance to Rift Valley Fever Virus (RVFV). These studies will build upon technological advances that permit us to find mutations faster than ever before, using massively parallel sequencing, both by itself and in combination with inducible pluripotent stem cell (IPS) technology in screens for cell-autonomous resistance to RVFV in mouse embryonic fibroblasts; MEFs. We have found that it is possible to screen mutagenized MEFs from reprogrammable C57BL/6J mice for resistance to RVFV ex vivo, to identify resistant clones, to use these clones to regenerate live mice, and at the same time, detect most of the mutations in these cells by sequencing genomic DNA and/or whole exomes. We have determined, based on in silico simulations, that compound heterozygous null alleles at almost all loci will result from mutagenesis on the scale we contemplate. As such, we hope to enrich our understanding of what it takes to combat a model herpesvirus infection with strong relevance to a human disease (HCMV infection), and also, to determine the critical host proteins necessary for a Category A pathogen (RVFV) to proliferate in mammalian cells. This work, pursued in depth, will inform us of molecular targets for the treatment of bunyavirus infections in general. In a third, circumscribed specific aim, we will analyze a new mutation, identified in screening, that abolishes the responses of plasmacytoid dendritic cells (PDCs) to nucleic acids. This mutation, called feeble, affects a channel protein that acts in conjunction with the adaptor protein 3 (APS) complex to condition endosomes in PDCs (but not other cells), making them competent to signal via TLRs. Based on our studies to date, we infer the existence of an ARF1->AP3^Slc45a->TLR7/9 pathway essential for the support of type I interferon gene induction in PDCs. The further elucidation of this pathway will deepen understanding of PDC function, and the role of PDC- derived interferon both in infection and in pathological settings such as systemic lupus erythematosus.

我们将扩展我们成功的小鼠巨细胞病毒（MCMV）易感性的正向遗传分析 感染，并建立一个新的屏幕上的细胞自主抵抗裂谷热病毒（RVFV）。 这些研究将建立在技术进步的基础上，使我们能够比以往任何时候都更快地发现突变， 使用大规模平行测序，通过自身和与诱导性多能干细胞组合， (IPS)在小鼠胚胎成纤维细胞中筛选对RVFV的细胞自主抗性的技术; MEFs。 我们已经发现，从可重编程的C57 BL/6 J小鼠中筛选诱变的MEF是可能的， 体外抗RVFV，鉴定抗性克隆，使用这些克隆再生活小鼠，以及 同时，通过对基因组DNA和/或整个细胞进行测序， 外显子组我们已经确定，基于计算机模拟，复合杂合无效等位基因， 几乎所有的基因座都是由我们预期规模的诱变产生的。因此，我们希望丰富我们的 了解如何对抗与人类密切相关的模型疱疹病毒感染 疾病（HCMV感染），并且还确定A类病毒感染所必需的关键宿主蛋白质。 病原体（RVFV）在哺乳动物细胞中增殖。这项工作，深入进行，将告诉我们分子 一般用于治疗布尼亚病毒感染的靶标。在第三个明确的目标中，我们将 分析一种新的突变，在筛选中发现，消除浆细胞样树突状细胞的反应 （PDCs）到核酸。这种被称为“虚弱”的突变会影响一种通道蛋白， 衔接蛋白3（APS）复合物调节PDCs（而不是其他细胞）内体，使它们 能够通过TLR发出信号。根据我们迄今为止的研究，我们推断 ARF 1-> AP 3-Slc 45 a-> TLR 7/9通路对于支持PDC中I型干扰素基因诱导至关重要。 对这一通路的进一步阐明将加深对PDC功能的理解，以及PDC在肿瘤发生中的作用。 在感染和病理环境如系统性红斑狼疮中衍生的干扰素。