Characterization of HCMV UL84

HCMV UL84 的表征

• 批准号: 7340542
• 负责人: GREGORY S PARI
• 金额: $ 30.35万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7340542
• 关键词: Amino Acid Sequence; Amino Acids; Bacteria; Binding; Binding Sites; Biological Assay; Boxing; Category Rating Scale; Cells; Class; Consensus; Cytomegalovirus; DNA; DNA Primase; DNA biosynthesis; DNA chemical synthesis; DNA replication origin; EMSA; Elements; Enzymes; Family; Fibroblasts; Genetic Transcription; Growth; Human; Immediate-Early Proteins; Kinetics; Late Promoters; Luc Gene; Luciferases; Lytic; Maps; Mediating; Metabolism; Nuclear; Open Reading Frames; Peptides; Phosphopeptides; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Polymerase; Protein Sequence Analysis; Proteins; RNA; RNA Helicase; Recombinants; Regulation; Reporter; Research Personnel; Response Elements; Role; SS DNA BP; Simian virus 40; System; Testing; Transcript; Transcription Coactivator; Transcriptional Activation; Transfection; Vero Cells; Viral; Viral Genome; Virus Diseases; base; chromatin immunoprecipitation; helicase; in vitro Assay; member; mutant; nucleoside triphosphatase; programs; promoter; research study; uridine triphosphatase

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.

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 的独特区域。使用瞬时复制测定阐明了参与 DNA 复制的酶。基于该测定和后续实验，证明 oriLyt 依赖性 DNA 复制需要以下开放阅读框 (ORF)：“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（立即早期蛋白）和 UL84（早期蛋白）。我们构建了 HCMV 重组 BAC，并在 UL84 基因座中插入。该 BAC 在 DNA 复制和将 UL44 和 IE2 正确分配到核复制区室方面都有缺陷。我们现已确定UL84在转染和感染细胞中被磷酸化，并且可以在转染细胞中与其自身形成稳定的物理结合。此外，UL84 具有 UTPase 活性，这可能是 DNA 复制中解旋酶活性能量产生系统的一部分。 UL84 的氨基酸序列分析表明，它是一个含有具有 RNA 解旋酶共有结构域的蛋白质的 DExD/H 盒。瞬时报告基因检测（其中 oriLyt 的一个区域连接到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，IE2 明显抑制了该启动子的高基础激活水平。然而，在许可型人成纤维细胞 (HFF) 中，oriLyt 启动子处于静止状态，并通过病毒感染或 UL84 和 IE2 转染而激活。瞬时测定还表明，可以通过整个复制机器的共转染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，并且 SV40 启动子可以在功能上替代这种活性。该提案将寻求 i) 确定 UL84 内的精确磷酸化位点； ii) 确定 UL84 磷酸化在 DNA 复制中的作用以及 UL84 介导的 IE2 调节； iii) 表征任何 UL84 相关的酶活性。