Baracchini E, Glezer E, Fish K, Stenberg R M, Nelson J A, Ghazal P
Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Virology. 1992 Jun;188(2):518-29. doi: 10.1016/0042-6822(92)90506-k.
The major immediate-early promoter (MIEP) of human cytomegalovirus directs the expression of several differentially spliced and polyadenylated mRNAs. These mRNAs encode nuclear phosphorproteins (IE55, IE72, and IE86), which consist of common and unique amino acid sequences. To date, very little is known of the functional role of the 55-kDa (IE55) protein. Here we present evidence that the IE55 protein is a positive activator of the MIEP. In human fibroblast cells IE55 protein activated the MIEP between 10- and 30-fold. Fusion of IE55 to the GAL4 DNA binding domain resulted in a chimeric protein capable of trans-activating a reporter with GAL4 recognition sequences. These results strongly suggest that IE55 is a bona fide transcriptional activator protein. In addition, the IE55 protein was found not to act synergistically with the IE72 activator protein. The IE55 protein shares the same amino acid sequence as IE86 except for a 154-amino-acid deletion at the C-terminal end of the protein. These proteins were functionally antagonistic; IE55 relieved repression by IE86 and, conversely, IE86 negated IE55 activation. Mutagenesis of the MIEP revealed that the target sequence for activation by IE55 is different from the IE86 autorepressive response element. These experiments suggest that the mechanism of action of the IE55 and IE86 isoforms is distinct. Moreover, from these results it is apparent that the interplay of these factors might be critical in determining the level of HCMV replication in the host.
人类巨细胞病毒的主要立即早期启动子（MIEP）指导几种差异剪接和聚腺苷酸化的mRNA的表达。这些mRNA编码核磷蛋白（IE55、IE72和IE86），它们由共同和独特的氨基酸序列组成。迄今为止，对于55kDa（IE55）蛋白的功能作用知之甚少。在此，我们提供证据表明IE55蛋白是MIEP的正激活因子。在人成纤维细胞中，IE55蛋白将MIEP激活了10至30倍。将IE55与GAL4 DNA结合域融合产生了一种嵌合蛋白，该蛋白能够反式激活带有GAL4识别序列的报告基因。这些结果强烈表明IE55是一种真正的转录激活蛋白。此外，发现IE55蛋白与IE72激活蛋白没有协同作用。IE55蛋白与IE86具有相同的氨基酸序列，只是在该蛋白的C末端有一个154个氨基酸的缺失。这些蛋白在功能上是拮抗的；IE55缓解了IE86的抑制作用，反之亦然，IE86否定了IE55的激活作用。对MIEP进行诱变发现，IE55激活的靶序列与IE86自身抑制反应元件不同。这些实验表明IE55和IE86亚型的作用机制是不同的。此外，从这些结果可以明显看出，这些因子之间的相互作用可能对确定宿主中HCMV复制的水平至关重要。
