| Project/Area Number |
11670299
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Virology
|
|---|
| Research Institution | Institute of Tropical Medicine, Nagasaki University |
|---|
Principal Investigator |
IGARASHI Akira Nagasaki University Professor, 熱帯医学研究所, 教授 (40029773)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SHIMIZU Hiroyuki Institute of Infectious Diseases Senior Researcher, ウイルス第2部, 厚生技官 (90270644)
HASEBE Futoshi Nagasaki University Research Associate, 熱帯医学研究所, 助手 (20253693)
MORITA Kouichi Nagasaki University Associate Professor, 熱帯医学研究所, 講師 (40182240)
TADANO Masayuki Ryukyu University Assistant Professor, 医学部, 助教授 (80179712)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Keywords | JEV / RNA belicase / ATPase / NS3 protein / RNA belicase |
|---|
| Research Abstract |
The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA Helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with His-tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP This result indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3. Several amino acid substitutions were introduced at the DExH motif of JEV NS3, Asp-285 and Glue-286 were found essential for both ATPase and RNA helicase activities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gln, Ser, or Arg could partly substitute for it. However, any other mutation at His-288 completely disrupted the RNA helicase activity of JEV NS3. The results suggested that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and ATPase and helicase activities are separable enzymatic functions.
|
