Reverse Genetic Analysis of Antiviral Resistance Mechanisms

我们的主要目标是阐明哺乳动物体内宿主抗病毒先天免疫反应的机制。到 迄今为止，我们还不完全了解病毒在侵入哺乳动物宿主时是如何被识别的。我们有 广泛研究了视黄酸诱导基因-1（RIG-I）的细胞内RNA病毒受体的作用， RIG-I家族及其信号通路，以及RIG-I家族与其他模式的关系 识别受体，特别是Toll样受体，在病毒识别中的作用。诱导类型的受体 I IFN对病毒来源的细胞质DNA的应答尚未被鉴定。最近的研究表明， 膜运输以及小的细胞器如内质网和高尔基体， 对于先天免疫系统识别细胞内病毒DNA很重要。此外，转录和 转录后调节对于控制对病毒的初始应答是重要的。拟议 项目，我们将利用小鼠反向遗传学来了解基因的功能，这些基因可能是 参与抗病毒免疫，聚焦3大主题。首先，我们将尝试识别潜在的分子 参与细胞内DNA病毒感应和控制DNA受体和信号传导的运输 分子。这将通过表达克隆或酵母双杂交筛选来实现，并将最终在 产生缺乏筛选中鉴定的候选分子的小鼠。其次，我们已经确定 单个分子的空间和时间调节都对DNA的形成有重要的作用， 发信号。在这个新的建议中，我们将更多地关注抗病毒反应的细胞生物学方面。我们将 使用设备分析活细胞中潜在参与抗病毒宿主防御的分子的运输 例如结构化照明显微镜（SIM）/光激活定位显微镜（PALM）和单个 分子成像技术。我们还将研究免疫细胞的运动及其相互作用， 病毒感染后的组织。大坂大学免疫学前沿研究中心的目标是 将尖端成像技术整合到免疫学研究中。小鼠建立的突变小鼠 由布鲁斯Beutler博士的小组的前沿遗传学将被带到大坂，和特定分子的作用， 膜运输将通过我们的显微技术进行分析。第三，我们将产生基因敲除小鼠 靶向可能参与抗病毒药物的转录和转录后基因调控的基因 应答此外，我们将产生潜在参与先天性抗病毒免疫的基因敲除小鼠， 在小鼠或果蝇中通过foi^ard遗传学鉴定的反应。 相关性（参见说明）： 病毒感染在世界上是一个严重的威胁，例如相对常见的新菌株的爆发， 流感。该项目旨在了解抗病毒免疫反应的基本机制。这 该项目将有助于实施新的治疗方法来对付病毒性传染病。

Our broad goal is to clarify mechanisms of host anti-viral innate immune responses in mammals in vivo. To date, we do not fully understand how viruses are recognized when they invade the mammalian host. We have extensively studied the roles of intracellular RNA virus receptors ofthe Retinoic acid-inducible Gene-l (RIG-I) family and their signaling pathways, and the relationship between the RIG-I family and other pattern recognition receptors, notably Toll-like receptors, in the recognition of viruses. The receptor(s) that induce type I IFN in response to cytoplasmic DNA of viral origin have yet to be identified. Recent studies suggested that membrane trafficking as well as small organelle such as endoplasmic reticulum and Golgi apparatus are important for the intracellular viral DNA recognition by the innate immune system. Further, transcriptional and post-transcriptional regulation are important for controlling initial responses to viruses. In the proposed projects, we will exploit mouse reverse genetics to understand the functions of genes that are potentially involved in anti-viral immunity, focusing on 3 major topics. First, we will try to identify molecules potentially involved in intracellular DNA virus sensing and in the control of trafficking of DNA receptors and signaling molecules. This will be achieved by expression cloning or yeast two-hybrid screening, and will culminate in the generation of mice lacking the molecular candidates identified in screening. Secondly, we have determined that both spatial and temporal regulation of individual molecules contributes in an important way to DNA signaling. In this new proposal, we will more focus on the cell biological aspects in anti-viral responses. We will analyze trafficking of molecules potentially involved in antiviral host defense in living cells using equipment such as structured illumination microscopy (SIM)/photo activated localization microscopy (PALM) and single molecule imaging techniques. We will also study the movement of immune cells and their interactions in tissues after viral infection. The Immunology Frontier Research Center at Osaka University is aiming to integrate cutting-edge imaging techniques in immunology research. Mutant mice established by mouse forward genetics by Dr. Bruce Beutler's group will be brought to Osaka, and the role of specific molecules in membrane trafficking will be analyzed by our microscopic techniques. Third, we will generate knockout mice targeting genes potentially involved in transcriptional and post-transcriptional gene regulation of anti-viral responses. Additionally, we will generate knockout mice potentially involved in innate anti-viral immune responses identified by foi^ard genetics either in mice or in Drosophila. RELEVANCE (See instructions): Virus infection is a serious threat in the world as exemplified by relatively common outbreaks of new strains of influenza. This project is aimed at understanding basic mechanisms of antiviral immune responses. This project will help to implement novel therapeutic approaches against viral infectious diseases.