Wen G, Chen C, Luo X, Wang Y, Zhang C, Pan Z
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Arch Virol. 2007;152(8):1565-73. doi: 10.1007/s00705-007-0969-2. Epub 2007 Apr 20.
The nonstructural protein 3 (NS3) of members of the family Flaviviridae possesses multiple enzyme activities that are likely to be essential for viral replication. Here, we cloned and expressed full-length CSFV NS3 protein (NS3FL) and its N-terminal truncated version (ntNS3) in E. coli. NTPase activities of the purified NS3FL and ntNS3 proteins and their reaction conditions were investigated. The results showed that CSFV NS3FL and ntNS3 proteins contained a specific polynucleotide-stimulated NTPase acitivity. Characterization of ntNS3 NTPase activity showed that optimal reaction conditions with respect to pH, MgCl2 and monovalent cations were similar to those of bovine viral diarrhea virus (BVDV) and hepatitis C virus (HCV). Site-directed mutagenesis analysis demonstrated that the GxGK(232)T to GxGAT mutation in the conserved motif I abolished the NTPase activity of ntNS3, whereas substitution of TATPA(354) for TATPV in the motif III had no effect on the enzyme activity. Moreover, the kinetic properties (K(m) and k(cat)) of CSFV NS3 were more similar to those of BVDV. Our results provide insight into the structure-function relationship of CSFV NS3 and facilitate our understanding of its role in the replication cycle of CSFV.
黄病毒科成员的非结构蛋白3（NS3）具有多种酶活性，这些活性可能对病毒复制至关重要。在此，我们在大肠杆菌中克隆并表达了全长猪瘟病毒NS3蛋白（NS3FL）及其N端截短版本（ntNS3）。研究了纯化的NS3FL和ntNS3蛋白的NTPase活性及其反应条件。结果表明，猪瘟病毒NS3FL和ntNS3蛋白具有特定的多核苷酸刺激的NTPase活性。ntNS3 NTPase活性的表征表明，在pH、MgCl2和单价阳离子方面的最佳反应条件与牛病毒性腹泻病毒（BVDV）和丙型肝炎病毒（HCV）相似。定点诱变分析表明，保守基序I中的GxGK(232)T突变为GxGAT消除了ntNS3的NTPase活性，而基序III中TATPA(354)被TATPV取代对酶活性没有影响。此外，猪瘟病毒NS3的动力学特性（K(m)和k(cat)）与牛病毒性腹泻病毒的更相似。我们的结果为深入了解猪瘟病毒NS3的结构-功能关系提供了线索，并有助于我们理解其在猪瘟病毒复制周期中的作用。
