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，很明显，UL84已成为被提议参与病毒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研究人员证明，UL84是一种多功能磷酸化和泛素化蛋白，可显示核细胞质穿梭，与病毒蛋白IE2， UL44， UL83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UL84在感染细胞中的细胞和病毒结合伙伴。这些发现为了解UL84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UL84与细胞激酶酪蛋白激酶II （CK2）相互作用，UL84中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CK2无法与UL84相互作用。这些残基的突变使UL84无法补充orilyt依赖的DNA复制，这表明磷酸化是UL84复制功能的必要修饰。此外，我们还发现UL84与pol辅助蛋白UL44相互作用。UL84与UL44的结合表明，pol辅助蛋白可能作为UL84与oriLyt相互作用的辅助因子，并可能将UL44作为复制复合体的一部分招募到oriLyt。此外，我们现在表明，在没有C/EBP的情况下，UL84与oriLyt中CCAAT/增强子结合（C/EBP）转录因子结合位点相互作用，这些位点对于oriLyt依赖性DNA复制至关重要。这是在oriLyt中首次报道UL84的明确结合位点。我们还证明了UL84的核输出活性对于orilyt依赖性DNA复制和病毒基因组环境至关重要。这表明UL84与病毒和/或细胞mRNA相互作用，这种相互作用在病毒复制中起重要作用。使用UL84- rna下拉实验，我们已经确定了编码IRS1的病毒mRNA是与UL84相关的一个转录本。该提案将寻求确定泛素化和ck2介导的磷酸化的翻译后修饰的作用，并阐明与oriLyt中特定元件相互作用的一整套细胞/病毒因子。最后，我们将确定与UL84相互作用的特定RNA物种，以及核质穿梭如何促进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.