Ide Y, Tanimoto A, Sasaguri Y, Padmanabhan R
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421, USA.
Gene. 1997 Nov 12;201(1-2):151-8. doi: 10.1016/s0378-1119(97)00440-x.
Hepatitis C virus (HCV) has a positive-strand RNA genome that codes for a polyprotein precursor, which is processed co- and post-translationally by cellular and viral proteinases into three structural and at least six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists in two phosphorylated forms of 56-kDa and 58-kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from HeLa cells and hepatocellular carcinoma (HepG2) cells by co-immunoprecipitation and by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein produced in E. coli. This protein kinase could phosphorylate in vitro the native NS5A on serine residues, (GST)-NS5A, histone H1, and casein as substrates. In addition, the GST-NS5A was also phosphorylated in vitro by the cAMP-dependent protein kinase A-alpha catalytic subunit.
丙型肝炎病毒（HCV）具有正链RNA基因组，该基因组编码一种多蛋白前体，其在翻译过程中和翻译后由细胞和病毒蛋白酶加工成三种结构蛋白和至少六种非结构（NS）蛋白。在哺乳动物细胞中表达的NS5A蛋白以56 kDa和58 kDa两种磷酸化形式存在。在本研究中，我们通过共免疫沉淀以及与固定化谷胱甘肽-S-转移酶（GST）-NS5A融合蛋白（该融合蛋白由大肠杆菌产生）的亲和力，证明了NS5A与来自HeLa细胞和肝癌（HepG2）细胞的一种蛋白激酶之间存在稳定的关联。这种蛋白激酶能够在体外将天然NS5A的丝氨酸残基、（GST）-NS5A、组蛋白H1和酪蛋白作为底物进行磷酸化。此外，GST-NS5A在体外也被cAMP依赖性蛋白激酶A-α催化亚基磷酸化。
