| Project/Area Number |
59440095
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Kyoto University |
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Principal Investigator |
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| Project Period (FY) |
1984 – 1985
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| Project Status |
Completed (Fiscal Year 1985)
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| Budget Amount *help |
¥23,700,000 (Direct Cost: ¥23,700,000)
Fiscal Year 1985: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1984: ¥21,000,000 (Direct Cost: ¥21,000,000)
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| Keywords | Influenza virus / Infectious cell entry / Endocytosis / Acidification / Membrane fusion / Conformational change of protein / ヘマグルチニン |
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| Research Abstract |
Based on the low pH-induced fusion activity of influenza virus, we proposed and presented evidence for a new infectious cell entry mechanism for influenza virus. The main subjects of the present investigation are the uncoating site of the virus in the acidic compartments and conformational change of the isolated hemagglutinin under acidic condition.
1. Analysis of local enviroment of the influenza virus in the cell by using fluorescent microscope.
(1). Measurement of pH of the viral enviroment.
The virus or isolated hemagglutinin were labelled with fluorescein isothiocyanate( FITC ) and endocytozed by cultured MDCK cells. The pH of the viral enviroment was estimated by the ratio of fluorescence intensities excited at two different wavelengths.
(2). Measurement of transport of the virus into lysosomes.
The transport of the virus particles into lysosomes was assayed by the change in the fluorescenceintensitity of FITC-diphosphate which was newly synthesized by us and caused about a 20-fold increase in the fluorescence intensity after dephosphorylation by lysosomal acidic phosphatase.
(3). Assembling of hyper-sensitive TV camera and image processor.
We assembled these instruments with the florescent microscope and measured the pH of intracellular vescicles which contained the FITC-labelled hemagglutinin. These equipments were also used to analyze saltatory motion of endosomes and transport of the vescicles to lysosomal region.
By combining these experiments with those on viral replication, we concluded that influenza virus is uncoated in endosomes before reaching lysosomes.
2. Conformational change of hemagglutinin under acidic condition.
By measuring the fluorescence from tryptophan residues in the hemagglutinin, it was shown that low pH induced the conformational change of the molecule to insert a part of the peptide into hydrophobic region of phospholipid bilayer containing ganglioside.
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