| Budget Amount *help |
¥10,400,000 (Direct Cost: ¥10,400,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2000: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1998: ¥3,600,000 (Direct Cost: ¥3,600,000)
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| Research Abstract |
Influenza virus NS1 protein stimulates translation of some viral proteins in vivo, Interaction among the NS1 protein, host translational initiation factors, and 5'-UTR of viral mRNA may be involved in this regulation. The NS1 protein is a phosphoprotein, however, it is not clear whether the phosphorylation is required for this activity.
We have obtained some NS1 mutant viruses using our reverse genetic system. Characterization pf these viruses indicated that dl12, which deletes 12 residues near N terminus of the NS1 protein, showed temperature-sensitive phenotype and the translation of all viral proteins was interfered. N110 virus, which deletes 52% of the C-terminus of the NS1 protein, has defect in the translation of the late viral proteins. We have also analyzed the phosphorylation in the virus-infected cells by using several protein kinase inhibitors. However the phoshorylation of the NS1 protein was not significantly interfered in that system.
We have then analyzed the phosphorylati
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on of the NS1 protein in vitro using purified A-, C-, or G-kinases. The NS1 protein was obtained by expression from plasmid DNA in E. coli and followed by affinity purification. Translational regulation by the NS1 protein was analyzed using rabbit reticulocyte lysate system in vitro. In this experiment, mRNAs were purified from WSN virus-infected MDBK cells. The NS1 protein was phosphorylated by either A-, C-, or G-kinases in vitro. Unphosphorylated NS1 protein was shown to stimulate the translation of the M1 and NP proteins. In addition, phosphorylation of the NS1 protein significantly stimulated this activity, indicating that the phosphorylation of the NS1 is not essential but important for the regulation. Relatively lower effect was observed for the translation of the NS1 using this system. We have then analyzed the NS1 mutants. The N110 NS1 was shown to stimulate the translation in vitro. C190 NS1, which deletes N-terminal 40 residues, has defect in that activity. These data together indicates that N-terminal110 residues has the functional domain enough for this activity. Less
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