| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Interferon regulatory factor-3 (IRF-3) is expressed constitutively in cytoplasm of a variety of cells. Infection of virus or bacteria leads to phosphorylation of multiple serine and threonine residues in the C-terminal region of IRF-3, which results in nuclear translocation of IRF-3 and ternary complex formation with CBP/p300 to induce the interferon β (INFβ) genen expression. However, little is known about the protein kinase that plays a critical role in the activation of IRF-3. The aim of this study is to investigate the mechanism of the signal transduction on the activation of IRF-3 by identifying the IRF-3 kinase.
For the purpose of expression cloning, HeLa cells were transformed by a plasmid which directs the H-2K^k gene or the green fluorescent protein (GFP) gene under the control of the IFNβ promoter. Stimulation by dsRNA led to the activation of either reporter activity, however, the cells adapted severe cytotoxic effects by dsRNA. Thus, the established cell lines are not suitab
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le for screening. Then, we constructed an assay system to monitor nuclear translocation of IRF-3, which should occur in advance to the reporter gene activation. For this purpose, we transfected a plasmid expressing a fusion protein between IEF-3 and GFP (GFP-IRF3), then examined subcellular localization of the fusion protein after stimulation by dsRNA or lipopolysaccharides and infection by Newcastle disease virus (NDV). In HeLa cells, neither stimuli led to nuclear translocation of GFP-IRF3. Among a variety of cell lines tested, only HT1080 showed nuclear translocation of GFP-IRF3 upon dsRNA stimulation or NDV infection.l The translocation of GFP-IRF3 was observed immediately after 30 min post-stimulation, and continued at least up to 10 hours. After 24 hours, cells were dead in the case of NDV infection, but not by dsRNA stimulation. Therefore, it may be possible to screen signal transducing molecules by monitoring the changes in subcellular localization of GFP-IRF3 with a stable transformant of HT1080. Less
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