Human cytomegalovirus DNA replication requires transcriptional activation via an IE2- and UL84-responsive bidirectional promoter element within oriLyt
Human cytomegalovirus DNA replication requires transcriptional activation via an IE2- and UL84-responsive bidirectional promoter element within oriLyt
复制标题
DOI：
10.1128/jvi.78.21.11664-11677.2004
复制
发表时间：
2004-11-01
影响因子：
5.4
通讯作者：
Pari, GS
中科院分区：
文献类型：
--
作者：
Xu, YY;Cei, SA;Pari, GS
人巨细胞病毒（HCMV）裂解源（oriLyt）在人成纤维细胞中的扩增依赖于6个核心复制蛋白和UL84、IE2和UL36-38。使用端粒酶永生化的人成纤维细胞系（T-HFs）， orilyt依赖的DNA复制不再需要UL36-38的基因产物。为了确定IE2在人成纤维细胞DNA复制中的作用，我们检测了HCMV oriLyt中潜在的IE2结合位点。我们现在发现一个强双向启动子（oriLyt(PM)）（核苷酸91754至92030）位于先前确定的起源核心区域，是oriLyt有效扩增所必需的。我们确定了一个14bp的新DNA基序（oriLyt启动子激活元件）是oriLyt（PM）活性所必需的，该基序最初被鉴定为直接早期蛋白IE2的结合元件。在Vero细胞中，oriLyt（PM）被IE2强烈抑制，但被UL84重新激活。相反，将oriLyt（PM）转染到人成纤维细胞中导致启动子活性的基础水平非常低，在感染HCMV后显著上调。共转染实验表明，UL84与IE2一起转染可激活人成纤维细胞的oriLyt（PM）。在共转染表达整个复制复合体的一组质粒时观察到进一步的激活。通过用猴病毒40早期启动子替换oriLyt(PM)，在没有IE2的情况下，在人成纤维细胞中观察到有效的oriLyt扩增。然而，在这些条件下，oriLyt的扩增仍然需要UL84。这些结果表明，HCMV裂解复制的启动机制部分涉及转录激活。
Amplification of the human cytomegalovirus (HCMV) lytic origin (oriLyt) in human fibroblasts is dependent upon six core replication proteins and UL84, IE2, and UL36-38. Using a telomerase-immortalized human fibroblast cell line (T-HFs), oriLyt-dependent DNA replication no longer required the gene products of UL36-38. To determine the role of IE2 in DNA replication in human fibroblasts, we examined potential IE2-binding sites within HCMV oriLyt. We now show that a strong bidirectional promoter (oriLyt(PM)) (nucleotides 91754 to 92030) is located in the previously identified core region of the origin and is required for efficient amplification of oriLyt. It was determined that a 14-bp novel DNA motif (oriLyt promoter activation element), which was initially identified as a binding element for the immediate-early protein IE2, was essential for oriLyt(PM) activity. In Vero cells the oriLyt(PM) was constitutively active and strongly repressed by IE2, but it was reactivated by UL84. In contrast, transfection of the oriLyt(PM) into human fibroblasts resulted in a very low basal level of promoter activity that was dramatically up-regulated upon infection with HCMV. Cotransfection assays demonstrated that the transfection of UL84 along with IE2 transactivated the oriLyt(PM) in human fibroblasts. Further activation was observed upon cotransfection of the set of plasmids expressing the entire replication complex. Efficient oriLyt amplification in the absence of IE2 in human fibroblasts was observed by replacing the oriLyt(PM) with the simian virus 40 early promoter. Under these conditions, however, UL84 was still required for amplification of oriLyt. These results suggest that the mechanism of initiation of HCMV lytic replication in part involves transcriptional activation.