Purification and characterization of the protein kinase encoded by the UL13 gene of herpes simplex virus type 2
Purification and characterization of the protein kinase encoded by the UL13 gene of herpes simplex virus type 2
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DOI：
10.1006/viro.1997.8653
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发表时间：
1997-08-18
期刊：
影响因子：
3.7
通讯作者：
Nishiyama, Y
中科院分区：
文献类型：
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作者：
Daikoku, T;Shibata, S;Nishiyama, Y
由单纯疱疹病毒1型（HSV-1）和2型（HSV-2）的UL 13基因编码的蛋白质已被预测为蛋白激酶。为了鉴定UL 13基因产物，我们制备了抗His.Tag-HSV-1UL 13融合蛋白的兔多克隆抗血清。该抗体与大肠杆菌中表达的60-kDa UL 13融合蛋白特异性反应，并且还识别HSV-1和HSV-2感染细胞的核组分中的56-至67-kDa晚期蛋白。另一方面，当用HSV-1和HSV-2感染Vero细胞时，在感染后期诱导新的酪蛋白激酶活性。活性的诱导在HSV-2感染的细胞的核组分中是最突出的，因此我们使用酪蛋白作为外源底物通过连续柱层析（磷酸纤维素、DEAE-纤维素和羟基磷灰石）从核提取物中纯化蛋白激酶（PK）。酶的最终制备物含有表观分子量为56 kDa的单一主要蛋白质，其与UL 13抗血清特异性反应。PK活性是最佳的情况下，NaCl和在相对较高的pH值。酸性蛋白质，如酪蛋白和胰高血糖素被有效地磷酸化的PK。作为HSV-2 US 3 PK的最佳底物的碱性蛋白鱼精蛋白也未被可检测地磷酸化，但组蛋白是UL 13 PK的相对良好的底物。磷酸化氨基酸分析显示PK磷酸化丝氨酸和苏氨酸，但不磷酸化酪氨酸。此外，发现该酶对肝素（酪蛋白激酶II（CK III）的有效抑制剂）具有高度抗性，并且还对CK 1-7（CK I的合成抑制剂）具有抗性，但对双特异性槲皮素非常敏感。这些结果表明HSV-2 UL 13 PK具有不同于细胞CK I、CK II和由US 3基因编码的病毒PK的独特催化性质。我们还确定了HSV-2 UL 13基因的完整核苷酸序列。HSV-2和HSV-1 UL 13 PKs之间的氨基酸同源性为85.9%，并且在C-末端区域高度保守。(C)北京：科学出版社.
The proteins encoded by the UL13 genes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) have been predicted to be protein kinases. To identify the UL13 gene product, we have raised a rabbit polyclonal antiserum against a His.Tag-HSV-1 UL13 fusion protein. The antibody specifically reacted with the 60-kDa UL13 fusion protein expressed in Escherichia coli and also recognized 56- to 67-kDa late proteins in nuclear fractions of HSV-1- and HSV-2-infected cells. On the other hand, novel casein kinase activity was induced at the late stage of infection when Vero cells were infected with HSV-1 and HSV-2. The induction of the activity was most prominent in the nuclear fractions of HSV-2-infected cells and therefore we purified the protein kinase (PK) from the nuclear extracts by successive column chromatography (phosphocellulose, DEAE-cellulose, and hydroxyapatite] using casein as an exogenous substrate. The final preparation of the enzyme contained a single major protein with an apparent molecular weight of 56 kDa which was specifically reacted with the UL13 antiserum. The PK activity was optimal in the absence of NaCl and at relatively high pH. Acidic proteins such as casein and phosvitin were efficiently phosphorylated by the PK. A basic protein, protamine, which is the best substrate for the HSV-2 US3 PK, was nor detectably phosphorylated but histone was a relatively good substrate for the UL13 PK. Phosphoamino acid analysis revealed that the PK phosphorylated serine and threonine but not tyrosine. Moreover the enzyme was found to be highly resistant to heparin, a potent inhibitor of casein kinase II (CK III and also resistant to CK 1-7, a synthetic inhibitor of CK I, but very sensitive to a bioflavonoid quercetin. These results indicate that the HSV-2 UL13 PK had unique catalytic properties different from those of cellular CK I, CK II, and the viral PK encoded by the US3 gene. We have also determined the complete nucleotide sequence of the HSV-2 UL13 gene. The overall amino acid homology between the HSV-2 and HSV-1 UL13 PKs was 85.9% and the homology was highly conserved in the C-terminal region. (C) 1997 Academic Press.