Reverse Genetic Analysis of Antiviral Resistance Mechanisms

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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.