Reverse Genetic Analysis of Antiviral Resistance Mechanisms

抗病毒耐药机制的反向遗传分析

• 批准号: 7481090
• 负责人: Shizuo Akira
• 金额: $ 43.43万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7481090
• 关键词: Affect; Antiviral Agents; Antiviral Response; Antiviral resistance; Cause of Death; Cells; Data; Development; Drosophila genus; Family; Gene Targeting; Generations; Genes; Genetic; Goals; Homologous Gene; Host Defense; Host Defense Mechanism; Immune response; Interferon Type I; Interferons; Knockout Mice; Lead; Mammals; Methods; Microarray Analysis; Molecular; Mus; Mutant Strains Mice; Mutation; Natural Immunity; Newcastle disease virus; Pathway Analysis; Pathway interactions; Phenotype; Production; Receptor Signaling; Research; Role; Screening procedure; Signal Pathway; Signal Transduction; Signaling Molecule; System; Toll-Like Receptor Pathway; Toll-like receptors; Viral; Viral Vaccines; Virus; Virus Diseases; Yeasts; concept; cytokine; detector; expression cloning; genetic analysis; in vivo; microbial; mouse model; novel; paralogous gene; positional cloning; resistance mechanism; response; small molecule; viral detection; yeast two hybrid system

We do not yet fully understand how host cells recognize and respond to viral infections, and our broad goal is to1 clarify mechanisms of anti-viral innate immunity in mammals. We have previously studied microbial components recognized by Toll-like receptors (TLRs) and established mouse models lacking each TLR. These studies revealed that viruses are recognized by TLR-dependent and -independent mechanisms and that recognition leads to induction of type I interferons (IFNs), cytokines critical for antiviral host defense in mammals. Although TLR signaling pathways leading to IFN production have been extensively studied, the molecular mechanisms of TLR-independent viral detection have remained poorly understood. Recently, we have focused on a family of potential cytoplasmic viral detectors and their signaling intermediates. We also have reason to believe that IFN-independent anti-viral host defense mechanisms operate in mammals, although we do not know precisely what these mechanisms might entail. In the present proposal, we plan to utilize mouse reverse genetics (gene targeting) to generate and analyzing mutant mice lacking molecules that have a strong likelihood of participating in antiviral responses. Combining targeted mutations with one another and using classical methods of pathway analysis, we hope to understand the how the presence of viruses is sensed, how signals are initiated, and how they are transduced to yield a defensive response. First, we will continue our studies of potential intracellular viral detectors and their signaling molecules leading to the production of type I IFNs and proinflammatory cytokines. Second, we will search for new candidate molecules potentially involved in signaling pathways that lead from from cytoplasmic viral detectors to permit type I IFN production. Toward this end, we will use expression cloning and yeast two-hybrid screening. Third, we will try to understand and characterize novel anti-viral host defense mechanisms. To do so we will rely upon data from the forward genetic initiatives pursued in Project 1 and Project 2. We will generate mice with mutations in homologues of candidate molecules identified by Drosophila forward genetics conducted by the Strasbourg group. We will also target paralogues of molecules identified by mouse forward genetics conducted by the La Jolla group. By comparing the phenotypes of these mice with those bearing mutations affecting pathways serving the TLRs or cytoplasmic viral detectors, we will develop a comprehensive understanding of the host response. The successful attainment of such an understanding is enormously important, since viral infections are one of the most important causes of death worldwide. Potentially, the new concepts that we develop may be applied to the improvement of anti-viral vaccines and to the development of small molecules that enforce anti-viral responses through interaction with newly identified molecules.

我们尚未完全了解宿主细胞如何识别和应对病毒感染，我们的总体目标是 to1 阐明了哺乳动物抗病毒先天免疫的机制。我们之前研究过微生物 Toll 样受体 (TLR) 识别的成分并建立了缺乏每种 TLR 的小鼠模型。 这些研究表明，病毒是通过 TLR 依赖性和非 TLR 依赖性机制来识别的，并且 识别导致诱导 I 型干扰素 (IFN)，这是抗病毒宿主防御至关重要的细胞因子 哺乳动物。尽管导致 IFN 产生的 TLR 信号通路已被广泛研究，但 不依赖 TLR 的病毒检测的分子机制仍知之甚少。最近，我们 重点研究了一系列潜在的细胞质病毒检测器及其信号中间体。我们也 有理由相信独立于干扰素的抗病毒宿主防御机制在哺乳动物中发挥作用， 尽管我们并不确切知道这些机制可能意味着什么。在本提案中，我们计划 利用小鼠反向遗传学（基因靶向）来生成和分析缺乏分子的突变小鼠 很有可能参与抗病毒反应。将目标突变相互结合 并使用经典的途径分析方法，我们希望了解病毒的存在是如何发生的 感知、信号如何发起以及如何转换它们以产生防御反应。首先，我们将 继续我们对潜在的细胞内病毒检测器及其信号分子的研究，从而导致 I 型干扰素和促炎细胞因子的产生。其次，我们将寻找新的候选分子 可能参与从细胞质病毒检测器引导至允许 I 型 IFN 的信号传导途径 生产。为此，我们将使用表达克隆和酵母双杂交筛选。第三，我们会尝试 了解和表征新型抗病毒宿主防御机制。为此，我们将依赖来自 项目 1 和项目 2 中所追求的前向遗传举措。我们将产生具有以下突变的小鼠 斯特拉斯堡果蝇正向遗传学鉴定出候选分子的同源物 团体。我们还将针对 La 实验室进行的小鼠正向遗传学鉴定的分子旁系同源物 乔拉集团.通过将这些小鼠的表型与携带影响通路的突变的小鼠进行比较 服务于TLR或细胞质病毒检测器，我们将对宿主有全面的了解 回复。成功实现这种理解非常重要，因为病毒感染 是全世界最重要的死亡原因之一。我们开发的新概念可能会 可应用于抗病毒疫苗的改进以及增强抗病毒作用的小分子的开发 通过与新识别的分子相互作用产生抗病毒反应。