Characterization of HCMV UL84

描述(申请人提供)：人巨细胞病毒DNA复制起始于一个不同的区域，称为DNA复制的裂解起点或oriLyt。利用瞬时复制实验对参与DNA复制的酶进行了鉴定。根据本实验和随后的实验，已经证明依赖于oriLyt的DNA复制需要下列开放阅读框架(ORF)：核心复制蛋白：UL54(聚合酶)、UL44(聚合酶辅助蛋白)、UL70(启动酶)、UL105(解旋酶)、UL102(启动酶相关因子)、UL57(单链DNA结合蛋白)、IE2-580aa(即时早期蛋白)和UL84(早期蛋白)。我们构建了人巨细胞病毒UL84位点处插入的重组BAC。这种BAC在DNA复制和将UL44和IE2正确地划分为核复制间隔方面都是有缺陷的。我们现在已经确定UL84在转基因和感染细胞中是磷酸化的，并且可以在转基因细胞中与自己形成稳定的物理结合。此外，UL84具有UTPase活性，这可能是DNA复制中解旋酶活性的能量生成系统的一部分。氨基酸序列分析表明，UL84是一个含有RNA解旋酶共同域的蛋白质的DExD/H盒。瞬时报告分析表明，oriLyt基因的一个区域连接在荧光素酶基因的上游，证明了oriLyt中存在强大的启动子。在Vero细胞中，IE2明显抑制该启动子的高基础水平的激活。然而，在允许性人成纤维细胞(HFF)中，oriLyt启动子处于静止状态，并被病毒感染或UL84和IE2的转染激活。瞬时分析还表明，通过共转染整个复制机制可以实现进一步的激活。我们目前的研究表明，oriLyt的很大一部分通过转录激活促进DNA合成，而SV40启动子可以在功能上取代这一活性。这项建议将寻求：1)确定UL84中的准确磷酸化位置；2)确定UL84在DNA复制和UL84介导的IE2调节中的作用；以及3)表征任何与UL84相关的酶活性。

DESCRIPTION (provided by applicant): HCMV DNA replication initiates at a distinct region called the lytic origin of DNA replication or oriLyt. Enzymes involved in DNA replication were elucidated using the transient replication assay. Based on this assay and subsequent experiments, it was demonstrated that the following open reading frames (ORFs) were required for oriLyt-dependent DNA replication: The "core" replication proteins : UL54 (polymerase), UL44 (polymerase accessory protein), UL70 (primase), UL105 (helicase), UL102 (primase associated factor), UL57 (single-stranded DNA binding protein) and IE2-580aa (immediate early protein) and UL84 (early protein). We constructed an HCMV recombinant BAC with an insertion in the UL84 loci. This BAC was defective for both DNA replication and proper partitioning of UL44 and IE2 into nuclear replication compartments. We have now determined that UL84 is phosphorylated in transfected and infected cells and can form a stable physical association with itself in transfected cells. In addition, UL84 has UTPase activity which is likely part of an energy-generating system for helicase activity in DNA replication. Amino acid sequence analysis of UL84 reveals that it is a DExD/H box containing protein with RNA helicase consensus domains. Transient reporter assays, in which a region of oriLyt was ligated upstream of the luciferase gene, demonstrated the presence of strong promoter within oriLyt. In Vero cells, IE2 apparently represses a high basal level of activation from this promoter. However in permissive human fibroblasts (HFFs), the oriLyt promoter is quiescent and is activated by viral infection or the transfection of UL84 and IE2. Transient assays also show that further activation can be achieved by the cotransfection of the entire replication machinery. Our current studies demonstrate that a substantial portion of oriLyt facilitates DNA synthesis by transcriptional activation and the SV40 promoter can functionally substitute this activity. This proposal will seek to i) determine the precise phosphorylation sites within UL84; ii) identify the role of UL84 phosphorylation in DNA replication and the UL84-mediated regulation of IE2; and, iii) characterize any UL84-associated enzymatic activity.