Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation.
Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation.
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独立克隆的巨细胞病毒装配蛋白 An 和 Ac 的一半可以通过分子间互补恢复装配蛋白突变体的蛋白水解活性。
DOI：
10.1128/jvi.71.2.956-964.1997
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Gibson,W
中科院分区：
文献类型：
--
作者：
Hall,MR;Gibson,W
疱疹病毒编码一种重要的丝氨酸蛋白酶，称为集合素，负责在衣壳形成过程中裂解前体组装蛋白。在巨细胞病毒(CMV)中，集合素在分子中间附近的一个内部(I)位点进行自我蛋白分解。I-位裂解将酶转化为由An和Ac亚基组成的活性双链形式。我们最近发现，重组的An和Ac亚基可以在真核细胞内自发结合，产生活性的双链蛋白酶。这一发现表明，这些亚基能够独立地承担其正确的功能构象，并促使我们测试它们是否能够进行分子间互补。这是通过将非活性突变(点、缺失和插入)形式的组装蛋白与对应于突变的组装素结构域的野生型亚基(An或Ac)一起表达来完成的。这些实验结果表明，An和Ac都能挽救装配素突变体的酶活性。突变的装配蛋白的I位裂解发生在互补过程中，但不是绝对必需的，如非活性装配与不可切割的I位点的有效互补所表明的那样。我们还发现，分子间互补可以挽救不活跃的突变全长蛋白酶前体的活性，并且可以发生在不同种类的CMV之间(例如，人CMV亚单位可以挽救突变的猿猴CMV组装蛋白的活性)。这些结果表明，组装蛋白能够形成可能具有重要功能的活性多聚体结构。
Herpesviruses encode an essential serine proteinase called assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of assemblin mutants. I-site cleavage of the mutated assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV assemblin). These results indicate that assemblin is able to form active multimeric structures that may be of functional importance.