Reverse Genetic Analysis of Antiviral Resistance Mechanisms

抗病毒耐药机制的反向遗传分析

• 批准号: 8871658
• 负责人: Shizuo Akira
• 金额: $ 60.23万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8871658
• 关键词: Antiviral Agents; Antiviral Response; Antiviral resistance; Autophagocytosis; Biological; Cells; Collaborations; Communicable Diseases; Cryopreservation; DNA; DNA Viruses; DNA receptor; Dendritic Cells; Derivation procedure; Disease Outbreaks; Drosophila genus; Embryo; Endoplasmic Reticulum; Equipment; Family; Funding; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic; Goals; Golgi Apparatus; Host Defense; Image; Imaging Techniques; Immune; Immune response; Immune system; Immunity; Immunology; Individual; Influenza; Instruction; Interferon Type I; Invaded; Knockout Mice; Life; Lighting; Mammals; Medical center; Membrane Protein Traffic; Microscopic; Microscopy; Molecular; Movement; Mus; Mutant Strains Mice; Organelles; Pattern recognition receptor; Post-Transcriptional Regulation; Proteins; RNA; RNA Viruses; Receptor Signaling; Regulation; Research; Resistance; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Structural Models; Structure; Techniques; Tissues; Toll-like receptors; Transcriptional Regulation; Transmembrane Transport; Tretinoin; Universities; Viral; Viral Genes; Viral Pathogenesis; Virus; Virus Diseases; Virus Receptors; Yeasts; Zinc Fingers; cytokine; expression cloning; forward genetics; frontier; gene function; gene interaction; genetic analysis; in vivo; macrophage; mutant; novel therapeutic intervention; receptor; resistance mechanism; response; reverse genetics; screening; single molecule; trafficking; viral DNA; yeast two hybrid system

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.

我们的总体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入侵哺乳动物宿主时如何被识别。我们有 广泛研究了视黄酸诱导基因-1 (RIG-I) 家族的细胞内 RNA 病毒受体的作用及其信号通路，以及 RIG-I 家族与其他模式识别受体（特别是 Toll 样受体）在病毒识别中的关系。尚未鉴定出响应病毒来源的细胞质 DNA 诱导 I 型 IFN 的受体。最近的研究表明，膜运输以及内质网和高尔基体等小细胞器对于先天免疫系统识别细胞内病毒 DNA 非常重要。此外，转录和转录后调控对于控制对病毒的初始反应非常重要。在拟议的项目中，我们将利用小鼠反向遗传学来了解可能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 病毒传感以及控制 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，并将最终产生缺乏筛选中确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对 DNA 信号传导有重要贡献。在这项新提案中，我们将更加关注抗病毒反应中的细胞生物学方面。我们将 使用结构照明显微镜 (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。大阪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。 Bruce Beutler博士团队通过小鼠正向遗传学建立的突变小鼠将被带到大阪，并研究特定分子在其中的作用 将通过我们的显微技术来分析膜运输。第三，我们将产生针对可能参与抗病毒反应的转录和转录后基因调控的基因的敲除小鼠。此外，我们将产生可能参与通过正向遗传学在小鼠或果蝇中鉴定的先天抗病毒免疫反应的基因敲除小鼠。