| Project/Area Number |
09670307
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Virology
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| Research Institution | YAMAGATA UNIVERSITY |
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Principal Investigator |
HONGO Seiji Yamagata University School of Medicine, Department of Bacteriology, Associate Professor, 医学部, 助教授 (90229245)
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| Co-Investigator(Kenkyū-buntansha) |
SUGAWARA Kanetsu Yamagata University School of Medicine, Department of Bacteriology, Assistant, 医学部, 教務職員 (60110673)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Influenza C virus / M gene / CM2 / ion channel / amantadine / シグナルペプチダーゼ |
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| Research Abstract |
The mechanism by which CM2 is produced from unspliced mRNA of RNA segment 6(M gene) was investigated. The introduction of the mutations.into the putative three initiation codons located between nucleotides 732 and 749 demonstrated that none of the three AUG codons are used for the initiation of CM2 synthesis. We recently identified a 374-amino-acid protein (designated P42) which is translated from the unspliced mRNA.P42 is an integral membrane protein containig two internal hydrophobic domains, one of which (residues 241 to 252) is followed by two sequences (252 Ile-Thr-Ser and 257 Ala-Ser-Ala) favorable for cleavage by signal peptidase.
The elimination of the second recognition motif for signal peptidase prevented CM2 protein from expressing both in the transfected Gas cells and in the translation in vitro in the presence of microsomal membranes. From these results, we conclude that cleavage of P42 by signal peptidase after Ala residue 259 produces CM2 composed of C-terminal 115 amino acids and M1' composed of N-terminal 259 amino acids.
To demonstrate the ion channel activity of CM2, we expressed this protein in oocytes of Xenopus laevis and measured whole cell currents by a two-electrode voltage-clamp procedure. It was found that the inward currents were induced upon hyperpolarizing the oocyte membranes. The amplitude of the currents increased slowly with time during the hyperpolarizing pulse, and the currents showed inward rectification characteristics, suggesting that CM2 forms a voltage-activated ion channel. We also obtained data indicating that the CM2-associated ion channel is less sensitive to change in pH than the M2-associated ion channel, and is resistantto block by anti-influenza A virus drug amantadine hydrochloride.
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