Study on the RNA Helicase Activity of the Flavivirus NS3 Proteins

黄病毒NS3蛋白RNA解旋酶活性的研究

• 批准号: 11670299
• 负责人: IGARASHI Akira
• 金额: $ 2.3万
• 依托单位: Institute of Tropical Medicine, Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670299/
• 关键词: JEV; RNA belicase; ATPase; NS3 protein; RNA belicase

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.

日本脑炎病毒（JEV）的NS 3蛋白含有RNA解旋酶/NTR的典型基序，但该蛋白没有RNA解旋酶活性的报道。为了鉴定和表征JEV NS 3的RNA解旋酶活性，在大肠杆菌中表达并纯化了具有His标签的截短形式的蛋白。纯化的JEV NS 3蛋白显示RNA解旋酶活性，其依赖于二价阳离子和ATP。该结果表明，C-末端457个残基足以显示JEV NS 3的RNA解旋酶活性。在JEV NS 3的DExH基序上引入了几个氨基酸取代，发现Asp-285和Glue-286是ATP酶和RNA解旋酶活性所必需的。Cys-287对RNA解旋酶活性起关键作用，但对ATP酶活性无影响。对JEV NS 3的His-288位点进行突变，发现His是ATP酶活性最好的氨基酸，Ala、Gly、Asn、Gln、Ser或Arg可部分替代His-288位点，而其它位点的突变则完全破坏RNA解旋酶活性。结果表明，Cys-287和His-288是JEV NS 3 RNA解旋酶活性的必需残基，而ATP酶和解旋酶活性是可分离的酶功能。

项目成果

Pandey B.D. et al.: "Severity-related molecular differences among nineteen strains of dengue type 2 viruses"Microbiol.Immunol.. 44. 179-188 (2000)

潘迪 B.D.

Pandey B.D., Morita K., Hosebe F., Parquet M.C., Igarashi A: "Molecular evolution, distribution and genetic relationship among the dengue 2 viruses isolated from different clinical severity"Southeast Asian J.Trop Med Publ.Heath.. 33. 266-272 (2000)

Pandey B.D.、Morita K.、Hosebe F.、Parquet M.C.、Igarashi A：“从不同临床严重程度分离的登革热 2 病毒的分子进化、分布和遗传关系”东南亚 J.Trop Med Publ.Heath.. 33. 266

Corazon C.Buerano et al: "IgM-Capture ELISA of Serum Samples Collected from Filipino Dengue Patients"Southeast Asian J.Trop Med Publ.Health. 30. 524-529 (2000)

Corazon C.Buerano 等人：“从菲律宾登革热患者收集的血清样本的 IgM 捕获 ELISA”东南亚 J.Trop Med Publ.Health。

Andi Utama, Hiroyuki Shimizu, Futoshi Hasebe, Kouichi Morita, Akira Igarashi, IkuoShoji, Yoshiharu Matsuura, Masahiro Hatsu, Kazuhiro Takamizawa, Akio Hagiwara and Tatsuo Miyamura.: "Role of the DExH Motif of the Japanese Encephalitis Virus and Hepatittis

Andi Utama、Hiroyuki Shimizu、Futoshi Hasebe、Kouichi Morita、Akira Igarashi、IkuoShoji、Yoshiharu Matsuura、Masahiro Hatsu、Kazuhiro Takamizawa、Akio Hagiwara 和 Tatsuo Miyamura。：“DexH 基序在日本脑炎病毒和肝炎中的作用

Andi Utama et al: "Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein"FEBS Letters. 465. 74-78 (2000)

Andi Utama 等人：“日本脑炎病毒 NS3 蛋白 RNA 解旋酶活性的鉴定和表征”FEBS Letters。