Xiao Ming, Wang Yujing, Chen Jiakuan, Li Bo
Biology Department, College of Life and Environment Sciences, Shanghai Teachers' University, Shanghai 200234, China.
Virus Genes. 2003 Aug;27(1):67-74. doi: 10.1023/a:1025176503138.
The full-length NS5B protein, and the truncated NS5B proteins of classical swine fever virus (CSFV) resulted from deletion of 24, 36, 65 or 82, amino acid residues at the C terminal were expressed in Escherichia coli cells and purified with a C-terminal hexahistidine tag. In addition to the full-length NS5B protein, those truncated NS5B proteins with deletion of 24, 36, or 65 amino acid residues were demonstrated to have RNA-dependent RNA polymerase (RdRp) activity, which was not found in the truncated NS5B proteins with deletion of 82 amino acid residues. Analysis of the template specificity of CSFV RdRp was done containing the different NS5B proteins with RdRp activity. It was shown that the template specificity of the enzyme was not strict with NS5B proteins truncated, suggesting that the C terminal of CSFV NS5B protein was involved in the template specificity of the enzyme. Site-directed mutagenesis of and prediction of the secondary structure of 3' terminal sequence of the template indicated that the cytidines at 3' terminus and the correct secondary structure of the template were essential to initiation of RNA synthesis by RdRp. Oxidation of the hydroxyl groups of the RNA template revealed that both the de novo initiation mechanism and the template-priming mechanism preference might be employed by the CSFV RdRp.
将经典猪瘟病毒（CSFV）的全长NS5B蛋白以及因C末端缺失24、36、65或82个氨基酸残基而产生的截短型NS5B蛋白在大肠杆菌细胞中表达，并用C末端六组氨酸标签进行纯化。除全长NS5B蛋白外，那些缺失24、36或65个氨基酸残基的截短型NS5B蛋白被证明具有RNA依赖性RNA聚合酶（RdRp）活性，而在缺失82个氨基酸残基的截短型NS5B蛋白中未发现这种活性。对具有RdRp活性的不同NS5B蛋白进行了CSFV RdRp模板特异性分析。结果表明，该酶对截短的NS5B蛋白的模板特异性不严格，这表明CSFV NS5B蛋白的C末端参与了该酶的模板特异性。对模板3'末端序列进行定点诱变及二级结构预测表明，模板3'末端的胞嘧啶和正确的二级结构对RdRp启动RNA合成至关重要。RNA模板羟基的氧化表明，CSFV RdRp可能采用从头起始机制和模板引发机制。
