The EICP22 protein of equine herpesvirus 1 physically interacts with the immediate-early protein and with itself to form dimers and higher-order complexes
The EICP22 protein of equine herpesvirus 1 physically interacts with the immediate-early protein and with itself to form dimers and higher-order complexes
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DOI：
10.1128/jvi.74.3.1425-1435.2000
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发表时间：
2000-02-01
影响因子：
5.4
通讯作者：
O'Callaghan, DJ
中科院分区：
文献类型：
--
作者：
Derbigny, WA;Kim, SK;O'Callaghan, DJ
马疱疹病毒1型（EHV-1）的EICP 22蛋白（EICP 22 P）是一种早期蛋白，与其他EHV-1调节蛋白协同作用，反式激活早期和晚期病毒基因的表达。我们以前已经确定EICP 22 P作为一个辅助调节蛋白，具有增强反式激活特性和序列特异性DNA结合活性的EHV-1立即早期蛋白（IEP）的能力。在本研究中，我们确定EICP 22 P作为一种自缔合蛋白，能够在感染过程中形成二聚体和高阶复合物。酵母双杂交系统的研究也表明EICP 22 P和IEP之间发生物理相互作用，EICP 22 P自聚集。体外和体内免疫共沉淀实验和谷胱甘肽S-转移酶（GST）下拉研究的结果证实了EICP 22 P和IEP之间的直接蛋白-蛋白相互作用以及EICP 22 P的自身相互作用。用IEP和EICP 22 P特异性抗体通过激光扫描共聚焦显微镜对感染细胞进行分析，发现这些病毒调节蛋白在感染后早期共定位于细胞核中，并在核质内形成致密核结构的聚集体。在酵母双杂交和GST下拉实验中，用一组EICP 22 P缺失突变体进行的突变分析表明，位置124和143之间的氨基酸是介导EICP 22 P自身相互作用的关键结构域。使用GST-EICP 22 P缺失突变体文库进行的其他体外蛋白结合试验将EICP 22 P区域2（氨基酸[aa] 65至196）和区域3（aa 197至268）内的氨基酸定位确定为介导其与IEP相互作用的残基。
The EICP22 protein (EICP22P) of Equine herpesvirus 1 (EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.