Ahn J H, Chiou C J, Hayward G S
The Molecular Virology Laboratories, Department of Pharmacology, Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, WBSB 317, Baltimore, MD 21205, USA.
Gene. 1998 Mar 27;210(1):25-36. doi: 10.1016/s0378-1119(98)00056-0.
The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region. Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so. Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells. In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2. Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas in the previous in-vitro study with truncated bacterial GST fusion proteins, it was mapped to between codons 346 and 579. Transient co-transfection assays with deleted IE2 effector genes in Vero cells showed that the extra segment of IE2 between codons 313 and 346 is also required for both autoregulation and transactivation activity in mammalian cells. In a two-hybrid assay to study IE2 self-interations, we generated both GAL4 DNA-binding (DB) and activation domain (A)/IE2 fusion proteins and showed that IE2 could also dimerize or oligomerize through the C-terminus of the protein in yeast cells. Domains required for this interaction were all mapped to within the region between codons 388 and 542, which is coincident with the domain mapped previously for dimerization by co-translation and immunoprecipitation in vitro. Comparison of the domains of the IE2 protein required for CRS binding and dimerization in yeast suggests that these activities correlate precisely with requirements for the negative autoregulation function of the IE2 protein in mammalian cells.
人巨细胞病毒（HCMV）主要立即早期（MIE）基因编码的86 kDa IE2核磷蛋白，既作为病毒和细胞基因表达的非特异性反式激活因子，又作为靶向其自身启动子/增强子区域帽位点的顺式抑制序列（CRS）的特异性DNA结合蛋白。尽管已证明在细菌中产生的IE2蛋白可与14 bp回文CRS基序结合，且体外合成的IE2通过该蛋白保守的C末端在溶液中形成稳定的二聚体，但尚无直接证据表明细胞内哺乳动物形式的IE2也能如此。在此，我们表明完整的HCMV IE2蛋白在酵母细胞中既能与CRS DNA结合，又能形成二聚体。在单杂交检测系统中，酵母细胞中表达的GAL4/IE2融合蛋白仅当CRS位点位于GAL1最小启动子上游时才能激活靶标HIS3的表达，但在突变的CRS位点上则不能，这表明IE2需要序列特异性DNA结合。对IE2 C末端一半的一系列缺失和三氨基酸点突变进行检测，将酵母中DNA结合所需的结构域定位到密码子313和579之间的整个区域，而在先前对截短的细菌GST融合蛋白的体外研究中，该结构域被定位到密码子346和579之间。在Vero细胞中用缺失的IE2效应基因进行瞬时共转染检测表明，IE2在密码子313和346之间的额外片段对于哺乳动物细胞中的自动调节和反式激活活性也是必需的。在研究IE2自身相互作用的双杂交检测中，我们生成了GAL4 DNA结合（DB）和激活结构域（A）/IE2融合蛋白，并表明IE2在酵母细胞中也能通过该蛋白的C末端形成二聚体或寡聚体。这种相互作用所需的结构域都被定位到密码子388和542之间的区域，这与先前通过体外共翻译和免疫沉淀确定的二聚化结构域一致。酵母中CRS结合和二聚化所需的IE2蛋白结构域的比较表明，这些活性与哺乳动物细胞中IE2蛋白负向自动调节功能的要求精确相关。
