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-I（RIG-I）家族的细胞内RNA病毒受体及其信号传导途径的作用，以及RIG-I家族与其他模式识别受体（尤其是Toll样受体）在病毒识别中的关系。诱导I型IFN应答病毒来源的细胞质DNA的受体还有待鉴定。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是重要的细胞内病毒DNA识别的先天免疫系统。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。在拟议的项目中，我们将利用小鼠反向遗传学来了解潜在参与抗病毒免疫的基因的功能，重点关注3个主要主题。首先，我们将尝试识别可能参与细胞内DNA病毒传感和控制DNA受体和信号分子运输的分子。这将通过表达克隆或酵母双杂交筛选来实现，并且将最终产生缺乏筛选中鉴定的分子候选物的小鼠。其次，我们已经确定了单个分子的空间和时间调节都以重要的方式对DNA信号传导做出贡献。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 使用诸如结构化照明显微镜（SIM）/光激活定位显微镜（PALM）和单分子成像技术等设备分析活细胞中可能参与抗病毒宿主防御的分子的运输。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旨在将尖端成像技术整合到免疫学研究中。由布鲁斯Beutler博士的小组通过小鼠正向遗传学建立的突变小鼠将被带到大坂，并研究特定分子在 膜运输将通过我们的显微技术进行分析。第三，我们将产生敲除小鼠靶基因可能参与转录和转录后基因调控的抗病毒反应。此外，我们将在小鼠或果蝇中产生通过正向遗传学鉴定的潜在参与先天性抗病毒免疫应答的敲除小鼠。

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 of the 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 forward genetics either in mice or in Drosophila.