| Summary: | 碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 99 === In recent years, RIG-I-like receptors (retinoic-acid-inducible gene I-like receptors, RLRs), have been identified that can recognize viral RNA in the cytoplasm and induce the production of type I interferon (IFN). We have established a high-throughput screening system to discover novel proteins that may be involved in the RLR signaling pathway and selected 15 potential candidates which seemed to affect anti-viral response. We first examined the roles of these candidates in RLR pathway by a reporter assay. The results showed that overexpression of proto-oncogene serine/threonine-protein kinase Pim-1 (Pim-1) enhanced the activation of IFN-β promoter induced by N-RIG (the N-terminal region of RIG-I) in a dose-dependent manner, whereas Pim-1 knockdown reduced the activation. We also examined the effects of Pim-1 on the phosphorylation of endogenous interferon regulatory factor 3 (IRF3) and the expression of IRF3 downstream gene (such as IFN-β, Interferon gamma-induced protein 10 kDa (IP10) and regulated upon activation, normal T-cell expressed, and secreted (RANTES)). All these data conclude that Pim-1 act as a positive regulator for the RIG-I-mediated signaling pathway. Since Pim-1 is a serine/threonine kinase, we therefore generated a kinase dead Pim-1, Pim-1(K67M), and further showed that the mutant Pim-1 significantly lost its regulatory effects, suggesting that Pim-1 regulated RLR signaling through it kinase activity. While Pim-1 strongly enhanced the activation of IFN-β promoter induced by N-RIG, Mitochondrial Antiviral Signaling protein (MAVS) or TANK binding kinase 1 (TBK1), it did not affect the activation of IFN-β promoter induced by Melanoma differentiation-associated gene 5 (MDA5), IRF3-5D (the constitutively active form of IRF3), Inhibitor of nuclear factor kappa-B kinase-beta (IKK-β) or Nuclear factor kappa B (NF-κB(p65)). The results suggest that Pim-1 may up-regulate RLR signaling pathway at a step upstream of MAVS but downstream of RIG-I. Co-immunoprecipitation experiment revealed that Pim-1 could associate with the N-terminal region of RIG-I and extended the half-life of the N-RIG protein. Collectively, these data demonstrate that Pim-1 up-regulates RIG-I-mediated antiviral responses by stabilizing the RIG-I protein through it kinase activity.
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