PA subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators
PA subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators
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DOI：
10.1128/jvi.75.18.8597-8604.2001
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发表时间：
2001-09-01
影响因子：
5.4
通讯作者：
Nieto, A
中科院分区：
文献类型：
--
作者：
Huarte, M;Sanz-Ezquerro, JJ;Nieto, A
流感病毒聚合酶复合体的PA亚基是一种磷酸化蛋白，可诱导共表达蛋白的蛋白水解降解。蛋白水解诱导减少的点突变体重建了复制缺陷但转录活性没有缺陷的病毒核糖核蛋白。为了寻找可能与PA蛋白相关的细胞因子，我们进行了酵母双杂交筛选。利用人类肾脏cDNA文库，我们鉴定了两个不同的相互作用克隆。其中一个被鉴定为先前描述的Gallus Gallus cDNA克隆CLE的人类同源物。人类基因编码36 kDa的蛋白质（hCLE），并在所有人体器官中普遍表达。通过拉下实验和辣椒斑点实验，观察PA和hCLE与纯化蛋白的相互作用。相互作用图谱显示，hCLE在PA蛋白序列的两个区域（位置493 ~ 512和557 ~ 574）与PA亚基相互作用。免疫荧光研究表明，hCLE蛋白定位于细胞核和细胞质中，尽管主要分布在细胞质中。hCLE被发现与活性的、高纯度的病毒核糖核蛋白相关，这些核糖核蛋白是由克隆的cdna在体内重组的，这表明PA-hCLE相互作用具有功能相关性。在数据库中搜索发现，hCLE与酵母因子Cdc68的中心区域有38%的序列同源性，该区域通过与反激活子相互作用来调节转录。与Cdc68转录激活因子同源家族的其他成员发现了类似的同源性，包括人类FACT蛋白。
The PA subunit of the influenza virus polymerase complex is a phosphoprotein that induces proteolytic degradation of coexpressed proteins. Point mutants with reduced proteolysis induction reconstitute viral ribonucleoproteins defective in replication but not in transcriptional activity. To look for cellular factors that could associate with PA protein, we have carried out a yeast two-hybrid screen. Using a human kidney cDNA library, we identified two different interacting clones. One of them was identified as the human homologue of a previously described cDNA clone from Gallus gallus called CLE. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiquitously in all human organs tested. The interaction of PA and hCLE was also observed with purified proteins in vitro by using pull-down and pep-spot experiments. Mapping of the interaction showed that hCLE interacts with PA subunit at two regions (positions 493 to 512 and 557 to 574) in the PA protein sequence. Immunofluorescence studies showed that the hCLE protein localizes in both the nucleus and the cytosol, although with a predominantly cytosolic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggesting that PA-hCLE interaction is functionally relevant. Searches in the databases showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with transactivators. Similar homologies were found with the other members of the Cdc68 homologue family of transcriptional activators, including the human FACT protein.