Identification of a promoter-specific transactivation domain in the herpes simplex virus regulatory protein ICP4.
Identification of a promoter-specific transactivation domain in the herpes simplex virus regulatory protein ICP4.
复制标题
单纯疱疹病毒调节蛋白 ICP4 中启动子特异性反式激活结构域的鉴定。
DOI：
10.1128/jvi.71.3.1757-1765.1997
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Wilcox,KW
中科院分区：
文献类型：
--
作者：
Xiao,W;Pizer,LI;Wilcox,KW
ICP 4在单纯疱疹病毒（HSV）感染的即刻早期阶段表达，并在随后的生产性感染阶段激活病毒基因的转录。α-疱疹病毒家族的几个成员编码与ICP 4具有广泛同源性的调节蛋白，并在N末端显示出反式激活结构域（transactivation domain，ICP 4）。ICP 4的核定位、DNA结合和二聚化所需的部分已经确定，但反式激活所需的特定结构域尚未确定。我们已经定义了一个启动子特异性的ICP 4酶的活性分析的GAL 4-ICP 4融合蛋白共转染到HeLa细胞与荧光素酶报告基因连接到一个启动子与5个GAL 4结合位点。GAL 4-ICP 4杂合体的反式激活活性完全位于ICP 4的前139个残基内，并且比源自ICP 4同源物的GAL 4-ICP 4杂合体的活性显著更弱。ICP 4残基97至109是该N-末端酶的关键组分。与HSV糖蛋白D（gD）或胸苷激酶（tk）启动子连接的ICP 4和荧光素酶基因的非融合形式进行的瞬时转染试验显示，ICP 4残基97至109是诱导gD启动子所需的，但不是诱导tk启动子所需的。与ICP 4同源物的比较实验显示，伪狂犬病病毒p53是gD启动子的有效激活剂和tk启动子的弱激活剂。互补分析显示，与野生型ICP 4相比，ICP 4残基97至109的缺失使来自感染细胞的病毒产量降低近500倍。我们得出结论，ICP 4残基97至109是一个启动子特异性的反式激活结构域，是所需的有效复制单纯疱疹病毒的核心组成部分。
ICP4 is expressed during the immediate-early phase of infection by herpes simplex virus (HSV) and activates transcription of viral genes during subsequent phases of productive infection. Several members of the alpha-herpesvirus family encode regulatory proteins that have extensive homology with ICP4 and exhibit a transactivation domain (TAD) at the N terminus. The portions of ICP4 required for nuclear localization, DNA binding, and dimerization have been defined, but a domain that is specifically required for transactivation has not been identified. We have defined a promoter-specific ICP4 TAD by analysis of the activity of GAL4-ICP4 fusion proteins cotransfected into HeLa cells with a luciferase reporter gene linked to a promoter with five GAL4 binding sites. The transactivation activity of GAL4-ICP4 hybrids is located entirely within the first 139 residues of ICP4 and is significantly less potent than the activity of GAL4-TAD hybrids derived from ICP4 homologs. ICP4 residues 97 to 109 are a critical component of this N-terminal TAD. Transient transfection assays performed with nonfusion forms of ICP4 and luciferase genes linked to the HSV glycoprotein D (gD) or thymidine kinase (tk) promoter revealed that ICP4 residues 97 to 109 are required for induction of the gD promoter but are not required for induction of the tk promoter. Comparative experiments with ICP4 homologs revealed that the pseudorabies virus TAD is a potent activator of the gD promoter and a weak activator of the tk promoter. Complementation assays revealed that loss of ICP4 residues 97 to 109 reduced the yield of virus from infected cells nearly 500-fold compared to wild-type ICP4. We conclude that ICP4 residues 97 to 109 are a core component of a promoter-specific transactivation domain that is required for efficient replication of herpes simplex virus.