Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16
Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16
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DOI：
10.1128/jvi.70.4.2124-2131.1996
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发表时间：
1996-04-01
影响因子：
5.4
通讯作者：
Capone, JP
中科院分区：
文献类型：
--
作者：
Schmelter, J;Knez, J;Capone, JP
单纯疱疹病毒反式激活因子VP16和病毒体宿主关闭蛋白vhs是病毒结构组分，其分别在感染期间指导立即早期基因表达的激活和宿主蛋白质合成的停滞。最近的研究表明，VP16和vhs在体外和感染细胞中相互作用，表明它们各自的调节功能是偶联的。在这份报告中，我们使用了酵母双杂交系统和亲和层析纯化的VP16融合蛋白，以精确地定位一个区域的vhs，指导与VP16的相互作用。vhs的缺失分析表明，一个21个氨基酸长的结构域跨越残基310至330（PAAGGTEMRVSWTEILTQQIA）是足以指导复合物的形成与VP16在体内和体外融合到一个异源蛋白。该区域的定点诱变鉴定出色氨酸321是体外和体内与VP16相互作用的关键决定因素，以及对体外稳定复合物形成重要的其他残基。这些发现表明，vhs残基310至330构成了一个独立的和模块化的结合界面，被VP16识别。
The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.