Puglielli M T, Woisetschlaeger M, Speck S H
Committee on Virology of Harvard University at Harvard Medical School, Boston, Massachusetts 02115, USA.
J Virol. 1996 Sep;70(9):5758-68. doi: 10.1128/JVI.70.9.5758-5768.1996.
During Epstein-Barr virus latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities are mutually exclusive. Upon infection, Wp is initially active, followed by a switch to Cp for the duration of latency. In this study, the region upstream of Cp was analyzed for the presence of cis elements involved in regulating the activities of the EBNA gene promoters in established in vitro immortalized lymphoblastoid cell lines (LCLs). It was determined that oriP, the origin for episomal maintenance during latency, is essential for efficient transcription initiation from either Cp or Wp in LCLs, as well as in some Burkitt's lymphoma cell lines. Deletion of the EBNA2-dependent enhancer located upstream of Cp resulted in a ca. two- to fivefold reduction in Cp activity in the LCLs assayed. More extensive deletion of sequences upstream of Cp, including the EBNA2-dependent enhancer, resulted in nearly complete loss of Cp activity. This loss of activity was shown to correlate with deletion of two CCAAT boxes, a proximal CCAAT box located at bp -61 to -65 and a distal CCAAT box located at bp -253 to -257, upstream of Cp. Site-directed mutagenesis of these cis elements demonstrated that Cp activity is highly dependent on the presence of a properly positioned CCAAT box, with the dependence on the distal CCAAT box apparent only when the proximal CCAAT box was deleted or mutated. Deletion of the glucocorticoid response elements located at ca. bp -850 upstream of Cp did not result in a significant loss in activity. In general, deletions which diminished Cp activity resulted in induction of Wp activity, consistent with suppression of Wp activity by transcriptional interference from Cp. The identification of oriP and the EBNA2-dependent enhancer as the major positive cis elements involved in regulating Cp activity in LCL suggests that EBNA gene transcription is largely autoregulated by EBNA 1 and EBNA 2.
在体外爱泼斯坦-巴尔病毒潜伏感染B淋巴细胞的过程中，六种病毒核抗原（EBNAs）从两个启动子之一，即Cp或Wp表达，这两个启动子的活性相互排斥。感染后，Wp最初是活跃的，随后在潜伏期间转换为Cp活跃。在本研究中，分析了Cp上游区域，以确定在已建立的体外永生化淋巴母细胞系（LCLs）中参与调节EBNA基因启动子活性的顺式元件。已确定oriP，即潜伏期间附加体维持的起源，对于LCLs以及一些伯基特淋巴瘤细胞系中从Cp或Wp进行有效的转录起始至关重要。删除位于Cp上游的EBNA2依赖性增强子导致所检测的LCLs中Cp活性约降低两到五倍。对Cp上游序列进行更广泛的删除，包括EBNA2依赖性增强子，导致Cp活性几乎完全丧失。这种活性丧失与位于Cp上游的两个CCAAT框的缺失相关，一个近端CCAAT框位于-61至-65碱基对处，一个远端CCAAT框位于-253至-257碱基对处。对这些顺式元件进行定点诱变表明，Cp活性高度依赖于一个位置合适的CCAAT框的存在，只有当近端CCAAT框被删除或突变时，对远端CCAAT框的依赖性才明显。删除位于Cp上游约-850碱基对处的糖皮质激素反应元件并未导致活性显著丧失。一般来说，降低Cp活性的缺失会导致Wp活性的诱导，这与Cp的转录干扰对Wp活性的抑制一致。将oriP和EBNA2依赖性增强子鉴定为参与调节LCL中Cp活性的主要正向顺式元件，表明EBNA基因转录在很大程度上由EBNA 1和EBNA 2自动调节。
