Ansari A, Emery V C
Department of Virology, Royal Free and University College Medical School, University College London, Royal Free Campus, Hampstead, London NW3 2PF, United Kingdom.
J Virol. 1999 Apr;73(4):3284-91. doi: 10.1128/JVI.73.4.3284-3291.1999.
The protein encoded by the U69 open reading frame (ORF) of human herpesvirus 6 (HHV-6) has been predicted to be a protein kinase. To investigate its functional properties, we have expressed the U69 ORFs from both HHV-6 variants, A and B, by using recombinant baculoviruses (BV6AU69 and BV6BU69). Nickel agarose and antibody affinity chromatography was used to purify the proteins to homogeneity and when incubated with [gamma-32P]ATP, both U69 proteins became phosphorylated on predominantly serine residues. These data strongly suggest that U69 is a protein kinase which autophosphorylates. The phosphorylation reaction was optimal at physiological pH and low NaCl concentrations. It required the presence of Mg2+ or Mn2+, and Mg2+ was able to support phosphorylation over a wider range of concentrations than Mn2+. Both ATP and GTP could donate phosphate in the protein kinase assay and the former was more efficient. U69 was capable of phosphorylating histone and casein (serine/threonine kinase substrates) but not enolase (a tyrosine kinase substrate). For the autophosphorylation reaction, the Michaelis constants for ATP of baculovirus-expressed HHV-6A and HHV-6B U69 were calculated to be 44 and 11 microM, respectively. U69 is a homologue of the UL97 gene encoded by human cytomegalovirus which has been shown to phosphorylate the antiviral drug ganciclovir (GCV). We analyzed whether the U69 ORF alone was capable of conferring GCV sensitivity on baculoviruses BV6AU69 and BV6BU69. In plaque reduction experiments, these baculoviruses displayed a GCV-sensitive phenotype compared to a control baculovirus (BVLacZ). The 50% inhibitory concentrations (IC50) of BV6AU69 and BV6BU69 were calculated to be 0.35 and 0.26 mM, respectively, whereas the control baculovirus had an IC50 of >1.4 mM. This shows that the U69 gene product is the only one required to confer GCV sensitivity on baculovirus.
人类疱疹病毒6型（HHV-6）的U69开放阅读框（ORF）编码的蛋白质被预测为一种蛋白激酶。为了研究其功能特性，我们通过使用重组杆状病毒（BV6AU69和BV6BU69）表达了HHV-6 A和B两个变体的U69 ORF。利用镍琼脂糖和抗体亲和层析将蛋白质纯化至同质，当与[γ-32P]ATP孵育时，两种U69蛋白主要在丝氨酸残基上发生磷酸化。这些数据有力地表明U69是一种能自身磷酸化的蛋白激酶。磷酸化反应在生理pH值和低NaCl浓度下最为适宜。它需要Mg2+或Mn2+的存在，并且Mg2+在比Mn2+更宽的浓度范围内都能支持磷酸化。在蛋白激酶测定中，ATP和GTP都能提供磷酸基团，且前者更有效。U69能够磷酸化组蛋白和酪蛋白（丝氨酸/苏氨酸激酶底物），但不能磷酸化烯醇化酶（酪氨酸激酶底物）。对于自身磷酸化反应，杆状病毒表达的HHV-6A和HHV-6B U69的ATP米氏常数分别计算为44和11微摩尔。U69是人类巨细胞病毒编码的UL97基因的同源物，已证明该基因能磷酸化抗病毒药物更昔洛韦（GCV）。我们分析了单独的U69 ORF是否能够赋予杆状病毒BV6AU69和BV6BU69对GCV的敏感性。在蚀斑减少实验中，与对照杆状病毒（BVLacZ）相比，这些杆状病毒表现出对GCV敏感的表型。BV6AU69和BV6BU69的50%抑制浓度（IC50）分别计算为0.35和0.26毫摩尔，而对照杆状病毒的IC50大于1.4毫摩尔。这表明U69基因产物是赋予杆状病毒对GCV敏感性所需的唯一物质。
