Reverse Genetic Analysis of Antiviral Resistance Mechanisms

抗病毒耐药机制的反向遗传分析

• 批准号: 7905006
• 负责人: Shizuo Akira
• 金额: $ 49.68万
• 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/7905006
• 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; Resistance; Role; Screening procedure; Signal Pathway; Signal Transduction; Signaling Molecule; System; Toll-Like Receptor Pathway; Toll-like receptors; Viral; Viral Vaccines; Virus; Virus Diseases; Yeasts; 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.

我们还不完全了解宿主细胞如何识别和响应病毒感染，我们的广泛目标是 阐明哺乳动物抗病毒先天免疫的机制。我们之前曾研究过微生物 Toll样受体(TLRs)识别的成分，以及缺乏TLR的已建立的小鼠模型。 这些研究表明，病毒是通过TLR依赖和独立的机制识别的 这种识别导致诱导I型干扰素(IFN)，这是抗病毒宿主防御的关键细胞因子 哺乳动物。尽管导致干扰素产生的TLR信号通路已经被广泛研究，但 TLR非依赖病毒检测的分子机制仍然知之甚少。最近，我们 已经集中在一系列潜在的细胞质病毒检测器及其信号中间体上。我们也 有理由相信不依赖干扰素的抗病毒宿主防御机制在哺乳动物中起作用， 尽管我们不确切地知道这些机制可能涉及到什么。在目前的建议中，我们计划 利用小鼠反向遗传学(基因打靶)产生和分析缺乏分子的突变小鼠 有很大的可能性参与抗病毒反应。将目标突变相互组合 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 感知，信号是如何启动的，以及它们是如何被传递以产生防御反应的。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。第二，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到允许I型干扰素的信号通路 制作。为此，我们将使用表达克隆和酵母双杂交筛选。第三，我们将努力 了解和表征新的抗病毒宿主防御机制。要做到这一点，我们将依靠来自 在项目1和项目2中推行的正向遗传计划。我们将在小鼠身上产生突变 由斯特拉斯堡进行的果蝇正向遗传学鉴定的候选分子的同系物 一群人。我们还将针对由洛杉矶大学进行的小鼠前向遗传学所识别的分子的类似物 乔拉集团。通过将这些小鼠的表型与那些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，我们将发展对宿主的全面了解 回应。成功地获得这样的理解是极其重要的，因为病毒感染 是全球最重要的死亡原因之一。潜在地，我们开发的新概念可能 应用于抗病毒疫苗的改进和小分子疫苗的开发 通过与新发现的分子相互作用而产生的抗病毒反应。