The regulatory role of cyclin-dependent protein kinases in human cytomegalovirus replication

人类巨细胞病毒（HCMV）的复制以病毒与宿主细胞紧密相互作用为特征，特别是细胞周期蛋白依赖性蛋白激酶（CDKs）在功能上被整合到有效的病毒基因表达和蛋白修饰中。CDK1、-2、-7和-9被确定为主要的hcmv调节激酶，尽管其潜在的调节机制仍在研究中。我们和其他人报道了CDK活性的药理学抑制诱导了病毒调节蛋白pUL69的核内聚集。相互作用分析显示pUL69与CDK/cyclin复合物之间存在蛋白关联。重要的是，我们证明了CDKs以及病毒蛋白激酶pUL97（最近被描述为病毒CDK同源物）具有特异性磷酸化这种病毒蛋白的能力。CDK抑制剂降低了pUL69已知的mRNA输出活性，这一发现表明了pUL69磷酸化的功能意义。这是第一个定义cdk和HCMV复制之间相互调节的特定接触点的例子。目前的项目将集中在CDK抑制剂诱导的核内聚集体形成的功能后果以及CDK- hcmv相互调节是否与这一发现有关的其他方面。在这种情况下，这是暗示更多的病毒蛋白参与病毒CDK相互作用，从初步结果得出结论。具体的实验将解决与CDK抑制剂诱导聚集体相关的异质蛋白复合物的组成和功能分析。此外，将产生突变病毒蛋白来解决磷酸化特征改变的问题以及cdk特异性磷酸化对这些病毒蛋白活性的影响。该研究将提供募集CDK活性作为HCMV复制内部途径的分子事件的详细描述。

Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction, as in particular cyclin-dependent protein kinases (CDKs) are functionally integrated into efficient viral gene expression and protein modification. CDK1, -2, -7 and -9 were identified as main HCMV-regulating kinases, albeit the underlying regulatory mechanisms are still under investigation. We and others reported that pharmacological inhibition of CDK activity induces intranuclear aggregation of the viral regulatory protein pUL69. Interaction analyses showed protein association between pUL69 and CDK/cyclin complexes. Importantly, we demonstrated that CDKs as well as the viral protein kinase pUL97, recently described as a viral CDK ortholog, possess the ability to specifically phosphorylate this viral protein. The functional significance of pUL69 phoshorylation was indicated by the finding that CDK inhibitors reduced its known mRNA export activity. This is the first example defining a specific contact point of the interregulation between CDKs and HCMV replication. The present project will concentrate on the question which functional consequences arise from the CDK inhibitor-induced intranuclear aggregate formation and whether there are further aspects of CDK-HCMV interregulation connected with this finding. In this context, it is suggestive that more viral proteins are involved in the viral CDK interaction, as concluded from preliminary results. Specific experiments will address the compository and functional analysis of heteromeric protein complexes which are associated with CDK inhibitor-induced aggregates. Moreover, mutant viral proteins will be generated to address the question of altered phosphorylation characteristics and the impact of CDK-specific phosphorylation on the activities of these viral proteins. The study will provide a detailed description of the molecular events which recruit CDK activities as an internal pathway of HCMV replication.