Expression, purification, and characterization of the RNA 5′-triphosphatase activity of dengue virus type 2 nonstructural protein 3
Expression, purification, and characterization of the RNA 5′-triphosphatase activity of dengue virus type 2 nonstructural protein 3
复制标题
DOI：
10.1006/viro.2002.1504
复制
发表时间：
2002-07-20
期刊：
影响因子：
3.7
通讯作者：
Padmanabhan, R
中科院分区：
文献类型：
--
作者：
Bartelma, G;Padmanabhan, R
登革病毒2型（DEN2）是黄病毒科正链RNA病毒的一员，包含一个在5'端具有I型帽结构的单RNA基因组。病毒RNA被翻译成一个单一的多蛋白前体，在被感染的宿主中加工成三种病毒粒子蛋白和至少七种非结构蛋白（NS）。NS3是一种多功能蛋白，在n端180个氨基酸残基内具有丝氨酸蛋白酶催化三联体，需要l作为蛋白酶活性激活的辅助因子。该催化三联体的c端部分具有在几种核苷三磷酸酶(NTPases)/RNA解旋酶中存在的保守基序。此外，来自感染细胞的枯草杆菌素处理的西尼罗河病毒NS3具有5'-RNA三磷酸酶活性，提示其在5'-帽结构的合成中起作用。在本研究中，用n端组氨酸标签在大肠杆菌中表达全长DEN2 NS3，并以可溶性形式纯化。纯化后的蛋白具有5'-RNA三磷酸酶活性，可切割5'-三磷酸化RNA底物的-磷酸部分。对NS3蛋白的生化和突变分析表明，NS3的核苷三磷酸酶和5′-RNA三磷酸酶活性具有共同的活性位点。(C) 2002 Elsevier Science （USA）。
Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5' end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires l as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5'-RNA triphosphatase activity, suggesting its role in the synthesis of the 5'-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5'-RNA triphosphatase activity that cleaves the gamma-phosphate moiety of the 5'-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside triphosphatase and 5'-RNA triphosphatase activities of NS3 share a common active site. (C) 2002 Elsevier Science (USA).