Characterization of HCMV UL84

描述（由申请人提供）：由于阐明了裂解DNA复制所需的病毒编码因子，因此很明显，UL 84已成为参与病毒DNA合成起始的调节和协调的中心因子。我们的实验室和其他实验室证明，UL 84是一种多功能磷酸化和泛素化的蛋白质，显示核质穿梭，与病毒蛋白IE 2，UL 44，UL 83相互作用，并结合RNA和DNA。我们实验室最近的蛋白质组学数据揭示了感染细胞中UL 84的细胞和病毒结合伴侣。这些发现现在为了解UL 84在HCMV生长中的确切作用开辟了新的研究领域。 我们现在已经证明了UL 84与细胞激酶酪蛋白激酶II（CK 2）相互作用，并且UL 84内的两个氨基酸残基介导了这种相互作用。这两个氨基酸残基（aa 148和157）的突变导致CK 2不能与UL 84相互作用。这些残基的突变使得UL 84不能补充oriLy依赖的DNA复制，这表明磷酸化是UL 84复制功能的必要修饰。此外，我们还发现UL 84与pol辅助蛋白UL 44相互作用。UL 84与UL 44的结合表明pol辅助蛋白可能作为UL 84与oriLyt相互作用的辅因子，并可能将UL 44募集到oriLyt作为复制复合物的一部分。此外，我们现在表明，UL 84与CCAAT/增强子结合（C/EBP）转录因子结合位点的oriLyt在C/EBP的情况下相互作用，这些网站是oriLyt依赖的DNA复制所必需的。这是首次报道在oriLyt中确定UL 84的结合位点。 我们还证明了UL 84的核输出活性对于oriLy依赖的DNA复制和病毒基因组的背景是必不可少的。这表明UL 84与病毒和/或细胞mRNA相互作用，并且这种相互作用在病毒复制中起重要作用。使用UL 84-RNA下拉测定，我们已经鉴定了编码IRS 1的病毒mRNA作为与UL 84相关的一种转录物。该提案将寻求确定泛素化和CK 2介导的磷酸化的翻译后修饰的作用，并阐明与oriLyt内的特定元素相互作用的完整的细胞/病毒因子。最后，我们将确定与UL 84相互作用的特定RNA种类以及核质穿梭如何有助于DNA合成的调节。

DESCRIPTION (provided by applicant): Since the elucidation of the viral-encoded factors required for lytic DNA replication it is clear that UL84 has emerged as the central factor proposed to be involved in the regulation and coordination of initiation of viral DNA synthesis. Our laboratory and others demonstrated that UL84 is a multifunctional phosphorylated and ubiquinated protein that displays nucleocytoplasmic shuttling, interacts with viral proteins IE2, UL44, UL83 and binds to RNA and DNA. Recent proteomics data from our laboratory revealed the cellular and viral binding partners for UL84 in infected cells. These discoveries have now illuminated new areas of research with respect to understanding the precise role of UL84 in HCMV growth. We have now demonstrated that UL84 interacts with the cellular kinase Casein Kinase II (CK2) and two amino acid residues within the UL84 mediated this interaction. Mutation of these two amino acid residues (aa 148 and 157) results in the inability of CK2 to interact with UL84. Mutation of these residues renders UL84 incapable of complementing oriLyt-depended DNA replication, suggesting that phosphorylation is an essential modification for the replication function of UL84. Additionally, we also show that UL84 interacts with UL44, the pol accessory protein. The binding of UL84 with UL44 suggests that the pol accessory protein may act as a cofactor for the interaction of UL84 with oriLyt and possibly recruits UL44 to oriLyt as part of the replication complex. Additionally, we now show that UL84 interacts with CCAAT/enhancer binding (C/EBP) transcription factor-binding sites within oriLyt in the absence of C/EBP, and these sites are essential for oriLyt-dependent DNA replication. This is the first reporting of a defined binding site for UL84 in oriLyt. We also demonstrate that the nuclear export activity of UL84 is essential for oriLyt-dependent DNA replication and in the context of the virus genome. This suggests that UL84 interacts with viral and/or cellular mRNA and this interaction plays an essential role in virus replication. Using an UL84-RNA pull down assay we have identified the viral mRNA encoding IRS1 as one transcript associated with UL84. This proposal will seek to determine a role for the posttranslational modifications of ubiquitination and CK2-mediated phosphorylation and elucidate the complete set of cellular/viral factors interacting with specific elements within oriLyt. Lastly, we will determine specific RNA species interacting with UL84 and how nucleocytoplasmic shuttling contributes to the regulation of DNA synthesis.