Reverse Genetic Analysis of Antiviral Resistance Mechanisms

我们还没有完全了解宿主细胞如何识别和应对病毒感染，我们的广泛目标是 阐明哺乳动物抗病毒天然免疫的机制。我们以前研究过微生物 Toll样受体（TLR）识别的组分，并建立了缺乏每种TLR的小鼠模型。 这些研究表明，病毒通过TLR依赖性和非依赖性机制识别， 这种识别导致I型干扰素（IFN）的诱导，I型干扰素是抗病毒宿主防御的关键细胞因子， 哺乳动物尽管导致IFN产生的TLR信号传导途径已被广泛研究， TLR非依赖性病毒检测的分子机制仍然知之甚少。最近我们 已经集中在一个家庭的潜在的细胞质病毒检测器和它们的信号中间体。我们也 有理由相信IFN-非依赖性抗病毒宿主防御机制在哺乳动物中起作用， 虽然我们不知道这些机制可能会带来什么。在目前的建议中，我们计划 利用小鼠反向遗传学（基因靶向）来产生和分析缺乏分子的突变小鼠， 很有可能参与抗病毒反应。将靶向突变相互结合 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，以及它们是如何被转换以产生防御反应的。一是 继续研究潜在的细胞内病毒检测器及其信号分子， I型IFN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到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，我们将努力 了解和表征新型抗病毒宿主防御机制。为此，我们将依靠来自 项目1和项目2中的前瞻性遗传倡议。我们将培育出突变的小鼠， 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源物 组我们还将靶向由La Jolla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 反应成功地获得这样的理解是非常重要的，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增强免疫力的小分子的开发 通过与新鉴定的分子相互作用产生抗病毒反应。

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.