Characterization of HCMV UL84

描述（由申请人提供）：HCMV DNA复制起始于称为DNA复制裂解起点或oriLyt的不同区域。使用瞬时复制试验阐明了参与DNA复制的酶。基于该测定和随后的实验，证明了oriLy依赖性DNA复制需要以下开放阅读框（ORF）：UL54（聚合酶），UL 44（聚合酶辅助蛋白）、UL 70（引发酶）、UL 105（解旋酶）、UL 102（引发酶相关因子）、UL 57（单链DNA结合蛋白）和IE 2 - 580 aa（立即早期蛋白）和UL 84（早期蛋白）。我们构建了在UL 84位点插入的HCMV重组BAC。该BAC对于DNA复制和将UL 44和IE 2适当分配到核复制区室两者都是有缺陷的。我们现在已经确定UL 84在转染和感染的细胞中被磷酸化，并且可以在转染的细胞中与自身形成稳定的物理缔合。此外，UL 84具有UTR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。UL 84的氨基酸序列分析表明，它是一个含有DExD/H盒的蛋白质，具有RNA解旋酶共有结构域。瞬时报告基因测定，其中oriLyt的一个区域连接在荧光素酶基因的上游，证明了oriLyt内存在强启动子。在Vero细胞中，IE 2显然抑制了来自该启动子的高基础水平的激活。然而，在允许的人成纤维细胞（HFF）中，oriLyt启动子是静止的，并且通过病毒感染或UL 84和IE 2的转染而被激活。瞬时测定还表明，可以通过共转染整个复制机制来实现进一步的激活。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，SV 40启动子可以在功能上取代这种活性。该提案将寻求i）确定UL 84内的精确磷酸化位点; ii）鉴定UL 84磷酸化在DNA复制和UL 84介导的IE 2调节中的作用;以及iii）表征任何UL 84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.