Cox G J, Parker M D, Babiuk L A
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada.
Virology. 1991 Nov;185(1):509-12. doi: 10.1016/0042-6822(91)90810-x.
To investigate the nature of the bovine coronavirus (BCV) ns2 protein, the gene encoding this protein was cloned and was expressed as a beta-galactosidase fusion protein. Antiserum raised against this protein reacted specifically with BCV-infected fixed cells in indirect immunofluorescence microscopy and precipitated an in vitro synthesized product approximately 32-kDa in molecular weight and an equivalent protein from BCV-infected cells. The synthesis of ns2 was found to be similar to the structural proteins of BCV and pulse-chase experiments indicated that ns2 protein was stable and that it accumulated in BCV-infected cells. Synthesis of ns2 in the presence of [32P] orthophosphate revealed that it is a phosphoprotein. Phosphoamino acid analysis confirmed the phosphorylated nature of ns2 and identified serine and threonine as its phosphorylated amino acid residues. This is the first demonstration of a phosphorylated nonstructural protein in coronavirus-infected cells.
为了研究牛冠状病毒（BCV）ns2蛋白的性质，编码该蛋白的基因被克隆并表达为β-半乳糖苷酶融合蛋白。用该蛋白制备的抗血清在间接免疫荧光显微镜下与BCV感染的固定细胞发生特异性反应，并沉淀出一种体外合成的分子量约为32 kDa的产物以及来自BCV感染细胞的同等蛋白。发现ns2的合成与BCV的结构蛋白相似，脉冲追踪实验表明ns2蛋白稳定且在BCV感染的细胞中积累。在[32P]正磷酸盐存在的情况下合成ns2表明它是一种磷蛋白。磷酸氨基酸分析证实了ns2的磷酸化性质，并确定丝氨酸和苏氨酸是其磷酸化氨基酸残基。这是首次在冠状病毒感染的细胞中证明存在磷酸化的非结构蛋白。
