Phosphorylation site mutations affect herpes simplex virus type IICP0 function
Phosphorylation site mutations affect herpes simplex virus type IICP0 function
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DOI：
10.1128/jvi.79.2.1232-1243.2005
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发表时间：
2005-01-01
影响因子：
5.4
通讯作者：
Schaffer, PA
中科院分区：
文献类型：
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作者：
Davido, DJ;von Zagorski, WF;Schaffer, PA
单纯疱疹病毒1型（HSV-1）的立即早期（IE）调节蛋白感染细胞蛋白0（ICP 0）是病毒和细胞基因两者的强的和全局的反式激活因子。在以前的研究中，我们报道了ICP 0是高度磷酸化的，并且包含至少七个不同的磷酸化信号，如通过磷酸胰蛋白酶肽图谱所确定的（D。J. Davido等人，J. Virol. 76：1077-1088，2002）。由于磷酸化影响许多病毒调节蛋白的活性，我们试图确定ICP 0的磷酸化是否影响其功能。为了解决这个问题，首先需要确定磷酸化的ICP 0区域。为了这个目的，ICP 0部分纯化，磷酸化位点的微毛细管高压液相色谱串联质谱进行了映射。三个磷酸化的区域含有11个假定的磷酸化位点，所有内或邻近的重要的反式激活活性的ICP 0的域，被确定。通过定点诱变将11个位点突变为丙氨酸，在三个区域的每一个中作为簇，产生表达ICP 0的突变形式的质粒：Phos I（四个突变位点）、Phos 2（三个突变位点）和Phos 3（四个突变位点）。一维磷酸胰蛋白酶肽分析证实，磷酸化状态的每一个磷酸盐突变形式的ICP 0的改变相对于野生型ICP 0。在功能测定中，ICP 0磷酸化位点突变影响ICP 0的亚细胞和亚核定位，其改变核结构域10（ND 10）相关蛋白PML染色模式的能力，和/或其在Vero细胞中的反式激活活性。然而，只有Phos 1的突变损害了ICP 0补充Vero细胞中ICP 0无效突变体复制的能力。因此，这项研究表明，磷酸化是ICP 0功能的重要调节因子。
The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos I (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.