Spatz S J, Nordby E C, Weber P C
Infectious Diseases Section, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
J Virol. 1996 Nov;70(11):7360-70. doi: 10.1128/JVI.70.11.7360-7370.1996.
The immediate-early protein ICP0 (infected-cell polypeptide 0) of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator of both viral and nonviral promoters in transient expression assays. Failure to splice the second of two introns in the ICP0 gene results in the utilization of an alternate stop codon that generates a truncated form of ICP0 called ICP0R. This protein exists in low levels in HSV-1-infected cells and functions as a dominant negative repressor of ICP0-mediated transactivation in transient expression assays. To conduct a detailed structure-function analysis of ICP0R, a series of insertion and deletion mutants of this protein were generated and analyzed in transfection assays. These studies indicated that segments of ICP0R that were rich in acidic amino acid residues (amino acids 9 to 76 and 233 to 241) or glycine residues (amino acids 242 to 262) were dispensable for the dominant negative phenotype. In contrast, the RING finger domain (amino acids 116 to 156) and surprisingly the sequences carboxy terminal to it (amino acids 157 to 232) were absolutely essential for transdominant repression. Consistent with these findings, the amino acid sequences of these two regions were conserved among other alphaherpesvirus ICP0 homologs. A construct containing only amino acids 76 to 232 inhibited ICP0-mediated transactivation almost as efficiently as wild-type ICP0R and represented the minimal sequences necessary for the dominant negative phenotype. These results demonstrated that the critical functional domain shared by both ICP0R and ICP0 is much more complex than a simple RING finger motif. Western blot (immunoblot) analyses of transfected cell lysates revealed that nearly all of the mutant constructs directed the expression of stable ICP0R proteins of the predicted molecular weight. However, there was a striking inverse correlation between the ability of a mutant construct to mediate transrepression and the amount of protein that it synthesized, indicating that dominant negative inhibition is achieved through the action of very little ICP0R protein.
单纯疱疹病毒1型（HSV-1）的立即早期蛋白ICP0（感染细胞多肽0）在瞬时表达试验中是病毒和非病毒启动子的一种多效性反式激活因子。未能剪接ICP0基因两个内含子中的第二个内含子会导致使用一个替代终止密码子，从而产生一种截短形式的ICP0，称为ICP0R。这种蛋白在HSV-1感染的细胞中水平较低，并且在瞬时表达试验中作为ICP0介导的反式激活的显性负性阻遏物发挥作用。为了对ICP0R进行详细的结构-功能分析，构建了该蛋白的一系列插入和缺失突变体，并在转染试验中进行分析。这些研究表明，富含酸性氨基酸残基（第9至76位氨基酸和第233至241位氨基酸）或甘氨酸残基（第242至262位氨基酸）的ICP0R片段对于显性负性表型是可有可无的。相反，环指结构域（第116至156位氨基酸）以及令人惊讶的是其羧基末端序列（第157至232位氨基酸）对于反式显性阻遏是绝对必需的。与这些发现一致，这两个区域的氨基酸序列在其他甲型疱疹病毒ICP-0同源物中是保守的。一个仅包含第76至232位氨基酸的构建体抑制ICP0介导的反式激活的效率几乎与野生型ICP0R相同，并且代表了显性负性表型所需的最小序列。这些结果表明，ICP0R和ICP0共有的关键功能结构域比简单的环指基序要复杂得多。对转染细胞裂解物的蛋白质印迹（免疫印迹）分析表明，几乎所有突变构建体都指导表达了预测分子量的稳定ICP0R蛋白。然而，突变构建体介导反式阻遏的能力与其合成的蛋白量之间存在显著的负相关，表明显性负性抑制是通过极少的ICP0R蛋白的作用实现的。
