Roberts M S, Boundy A, O'Hare P, Pizzorno M C, Ciufo D M, Hayward G S
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
J Virol. 1988 Nov;62(11):4307-20. doi: 10.1128/JVI.62.11.4307-4320.1988.
In transient-expression assays, the IE175 (alpha 4) promoter region of herpes simple virus is down-regulated after cotransfection with DNA encoding its own protein product (IE175 or ICP4). The inhibition by IE175 proved to be highly specific for its own promoter region and did not act on either the herpes simplex virus type 1 IE110 (alpha 0) or human cytomegalovirus major immediate-early promoters. Furthermore, the inhibition was still exhibited by IE175 effector plasmids driven by strong heterologous promoters and therefore must be a direct autoregulatory response that cannot be explained by promoter competition effects. In gel mobility retardation assays with infected-cell nuclear extracts, a prominent and specific DNA-protein complex was formed with DNA fragments containing sequences from -108 to +30 in the IE175 promoter region. This activity was not present in mock-infected samples. Even stronger binding occurred with a fragment containing sequences from -128 to +120 in the IE110 promoter, but this second locus was not associated with any detectable response phenotype in cotransfection assays. Supershift experiments with an anti-IE175 monoclonal antibody confirmed the presence of the IE175 protein in both DNA-protein complexes. In the IE175 promoter, specific binding correlated closely with the presence of an intact autoregulatory signal near the cap site as judged by the loss of both activities in a 3'-deleted promoter fragment lacking sequences from -7 to +30. Insertion of a cloned 30-mer synthetic oligonucleotide sequence from positions -8 to +18 in IE175 restored both IE175 binding activity and the down-regulation phenotype. Direct shift-up assays with a similar 30-base-pair (bp) oligonucleotide containing 21 bp from positions -75 to -55 of IE110 (which encompasses a consensus ATCGTC motif) also produced a specific DNA-protein complex containing the IE175 protein. This ATCGTC motif proved to be a necessary component of both the IE110 and IE175 binding sites, but was insufficient on its own for complex formation. Finally, deletion of 2 bp from positions -3 and -4 within the ATCGTC sequence in the IE175 cap site region abolished both binding activity and the IE175-dependent autoregulation phenotype.
在瞬时表达分析中，单纯疱疹病毒的IE175（α4）启动子区域在与编码其自身蛋白产物（IE175或ICP4）的DNA共转染后被下调。事实证明，IE175对其自身启动子区域的抑制具有高度特异性，对单纯疱疹病毒1型IE110（α0）或人巨细胞病毒主要立即早期启动子均无作用。此外，由强异源启动子驱动的IE175效应质粒仍表现出抑制作用，因此这一定是一种直接的自动调节反应，无法用启动子竞争效应来解释。在用感染细胞核提取物进行的凝胶迁移率阻滞分析中，在IE175启动子区域中含有从-108至+30序列的DNA片段形成了一个显著且特异的DNA-蛋白质复合物。在模拟感染的样品中不存在这种活性。在IE110启动子中，含有从-128至+120序列的片段产生了更强的结合，但在共转染分析中，这第二个位点与任何可检测到的反应表型均无关。用抗IE175单克隆抗体进行的超迁移实验证实了两种DNA-蛋白质复合物中均存在IE175蛋白。在IE175启动子中，特异性结合与帽位点附近完整自动调节信号的存在密切相关，这可通过在缺少从-7至+30序列的3'-缺失启动子片段中两种活性的丧失来判断。从IE175中-8至+18位置插入一个克隆的30聚体合成寡核苷酸序列可恢复IE175结合活性和下调表型。用一个类似的30碱基对（bp）寡核苷酸进行直接迁移增强分析，该寡核苷酸含有来自IE110的-75至-55位置的21 bp（其中包含一个共有ATCGTC基序），也产生了一个含有IE175蛋白的特异性DNA-蛋白质复合物。事实证明，这个ATCGTC基序是IE110和IE175结合位点的必要组成部分，但仅凭其自身不足以形成复合物。最后，在IE175帽位点区域的ATCGTC序列中从-3和-4位置缺失2 bp消除了结合活性和IE175依赖性自动调节表型。
