The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins
The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins
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DOI：
10.3390/v5123213
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发表时间：
2013-12-01
期刊：
影响因子：
4.7
通讯作者：
Marschall, Manfred
中科院分区：
文献类型：
--
作者：
Graf, Laura;Webel, Rike;Marschall, Manfred
人巨细胞病毒（HCMV）编码的蛋白激酶pUL 97，被认为是细胞周期蛋白依赖性激酶（CDK）的直系同源物，由于共同的结构和功能特征。CDK活化的主要机制是结合相应的细胞周期蛋白，包括细胞周期蛋白T1，它是CDK 9的常用调节辅因子。本研究利用酵母双杂交和免疫共沉淀技术证明了pUL 97和细胞周期蛋白T1之间的直接相互作用。共聚焦免疫荧光显示部分共定位的pUL 97与细胞周期蛋白T1在亚核区室，最明显的病毒复制中心。pUL 97和cyclin T1的分布模式与HCMV毒株和宿主细胞类型无关。负责与细胞周期蛋白T1相互作用的pUL 97的序列结构域在氨基酸231-280之间。另外的免疫共沉淀分析显示细胞周期蛋白B1和细胞周期蛋白A作为pUL 97的进一步相互作用伴侣。在ATP消耗试验中对pUL 97-细胞周期蛋白T1相互作用的研究强烈表明，pUL 97被CDK 9/细胞周期蛋白T1复合物以底物浓度依赖性方式磷酸化。这是疱疹病毒CDK直系同源物和细胞周期蛋白之间相互作用的第一个证明。
The human cytomegalovirus (HCMV)-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK) ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231-280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.