Study on the RNA Helicase Activity of the Flavivirus NS3 Proteins

日本脑炎病毒（JEV）的NS3蛋白含有典型的RNA解旋酶/NTPase基序，但尚未报道该蛋白具有RNA解旋酶活性。为了鉴定和表征JEV NS3的RNA解旋酶活性，我们在大肠杆菌中表达了带有His-tag的该蛋白的截断形式并进行了纯化。纯化得到的JEV NS3蛋白具有RNA解旋酶活性，其解旋酶活性依赖于二价阳离子和ATP，表明其c端457残基足以显示JEV NS3的RNA解旋酶活性。在JEV NS3的DExH基序上引入了几个氨基酸替换，发现Asp-285和Glue-286对atp酶和RNA解旋酶活性都是必需的。Cys-287对乙脑病毒NS3的RNA解旋酶活性至关重要，但对atp酶活性不重要。对乙脑病毒NS3的His-288位点进行突变，发现His是atp酶活性最优的氨基酸，Ala、Gly、Asn、Gln、Ser或Arg可以部分替代。然而，His-288的任何其他突变都完全破坏了JEV NS3的RNA解旋酶活性。结果表明，Cys-287和His-288是JEV NS3 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。

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.