Characterization of HCMV UL84

描述（由申请人提供）：HCMV DNA复制开始于一个称为DNA复制裂解起源或oriLyt的独特区域。参与DNA复制的酶被利用瞬时复制试验阐明。基于该分析和随后的实验，证明orilyt依赖性DNA复制需要以下开放阅读框（ORFs）：“核心”复制蛋白：UL54（聚合酶）、UL44（聚合酶辅助蛋白）、UL70（引物酶）、UL105（解旋酶）、UL102（引物酶相关因子）、UL57（单链DNA结合蛋白）和IE2-580aa（即时早期蛋白）和UL84（早期蛋白）。我们构建了一个在UL84位点插入的HCMV重组BAC。该BAC在DNA复制和UL44和IE2正确划分到核复制区室方面都存在缺陷。我们现在已经确定UL84在转染和感染的细胞中磷酸化，并且可以在转染的细胞中与自身形成稳定的物理关联。此外，UL84具有UTPase活性，这可能是DNA复制中解旋酶活性的能量产生系统的一部分。氨基酸序列分析表明UL84是一个含有RNA解旋酶一致结构域的DExD/H box蛋白。在瞬时报告基因分析中，oriLyt的一个区域连接在荧光素酶基因的上游，证明oriLyt中存在强启动子。在Vero细胞中，IE2明显抑制该启动子的高基础水平激活。然而，在允许的人成纤维细胞（HFFs）中，oriLyt启动子是静止的，并被病毒感染或UL84和IE2转染激活。瞬时实验也表明，进一步的激活可以通过整个复制机制的共转染来实现。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替代这种活性。本提案将寻求i)确定UL84中的精确磷酸化位点；ii)确定UL84磷酸化在DNA复制和UL84介导的IE2调控中的作用；iii)表征任何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.