Pizzorno M C, O'Hare P, Sha L, LaFemina R L, Hayward G S
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Virol. 1988 Apr;62(4):1167-79. doi: 10.1128/JVI.62.4.1167-1179.1988.
The major immediate-early (IE) gene region mapping at coordinates 0.71 to 0.74 in the genome of human cytomegalovirus (HCMV) gives rise to a series of overlapping spliced IE mRNAs that are all under the transcriptional control of the complex IE68 promoter-enhancer region. We show here that one of the phosphorylated nuclear proteins encoded by this region behaves as a powerful but nonspecific trans-activator of gene expression. In transient chloramphenicol acetyltransferase (CAT) assay experiments with Vero cells all relatively weak heterologous target promoters tested, including those of herpes simplex virus IE175 and delayed-early genes, adenovirus E3, the enhancerless simian virus 40 early gene, and the human beta interferon gene, were stimulated between 30- and 800-fold by cotransfection with the HindIII C fragment of HCMV (Towne) DNA. In contrast, expression of the homologous HCMV IE68-CAT gene but not SV2-CAT was specifically repressed. Inactivation mapping studies of the effector DNA, together with dose-response comparisons with subclones from the region, revealed that an intact 7.1-kilobase sequence encompassing both the IE1 and IE2 coding regions (exons 1 to 5) in the major IE transcription complex was required for both the nonspecific trans-activation and autoregulatory responses. The IE1 coding region alone (exons 1 to 4) was inactive, but both functions were restored by insertion of the IE2 coding region (exon 5) in the correct orientation downstream from the IE1 coding region. Internal deletions or inserted terminator codons in IE1 (exon 4) still gave efficient trans-activation and autoregulation, whereas the insertion of terminator codons in IE2 (exon 5) abolished both activities. Finally, IE2 (exon 5) sequences only (under the direct transcriptional control of the strong simian CMV IE94 promoter) were still able to specifically down regulate IE68-CAT expression but failed to exhibit trans-activation properties. Therefore, the IE2 gene product(s) of HCMV appear likely to be key control proteins involved in gene regulation during HCMV infection.
人巨细胞病毒（HCMV）基因组中位于坐标0.71至0.74处的主要立即早期（IE）基因区域产生了一系列重叠的剪接IE mRNA，这些mRNA均受复杂的IE68启动子-增强子区域的转录控制。我们在此表明，该区域编码的一种磷酸化核蛋白表现为基因表达的强大但非特异性反式激活因子。在对Vero细胞进行的瞬时氯霉素乙酰转移酶（CAT）分析实验中，所有测试的相对较弱的异源靶启动子，包括单纯疱疹病毒IE175和延迟早期基因、腺病毒E3、无增强子的猿猴病毒40早期基因以及人β干扰素基因的启动子，与HCMV（Towne）DNA的HindIII C片段共转染后，受到30至800倍的刺激。相反，同源的HCMV IE68 - CAT基因而非SV2 - CAT的表达受到特异性抑制。对效应DNA的失活图谱研究，以及与该区域亚克隆的剂量反应比较，揭示了主要IE转录复合物中包含IE1和IE2编码区（外显子1至5）的完整7.1千碱基序列对于非特异性反式激活和自调节反应都是必需的。单独的IE1编码区（外显子1至4）无活性，但通过将IE2编码区（外显子5）以正确方向插入IE1编码区下游，两种功能得以恢复。IE1（外显子4）中的内部缺失或插入终止密码子仍能产生有效的反式激活和自调节，而在IE2（外显子5）中插入终止密码子则消除了这两种活性。最后，仅IE2（外显子5）序列（在强猿猴CMV IE94启动子的直接转录控制下）仍能够特异性下调IE68 - CAT的表达，但未能表现出反式激活特性。因此，HCMV的IE2基因产物似乎可能是HCMV感染期间参与基因调控的关键控制蛋白。
