Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome
Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome
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DOI：
10.1074/jbc.274.24.17358
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发表时间：
1999-06-11
影响因子：
4.8
通讯作者：
Engelman, A
中科院分区：
文献类型：
--
作者：
Chen, HM;Wei, SQ;Engelman, A
采用Mu介导的聚合酶链反应足迹法研究人类免疫缺陷病毒I型（HIV-I）整合前复合物的蛋白质-DNA结构。整合前复合物部分纯化后，使用一个既定的共培养感染技术，以及一种新的技术，使用无细胞上清液从转染细胞作为病毒的来源。足迹法显示，结合蛋白保护每个病毒末端的末端200-250个碱基对免受核酸酶攻击。结合蛋白质也引起强烈的转座增强附近的每一个末端的HIV-1。相比之下，病毒DNA末端内部的区域没有显示出强蛋白结合的证据。整合前HIV-I的末端区域显然形成了独特的核蛋白结构，我们将其称为整合体，以将其与更大的整合前复合体区分开来。我们的新系统还使我们能够分析从整合酶突变病毒感染的细胞中分离的整合前复合物的结构和功能。复合物来源于整合酶催化、整合酶与病毒DNA底物结合或整合酶蛋白羧基末端结构域中未知功能缺陷的病毒。这些突变体复合物都不支持可检测的整合活性。尽管在纯化的样品中存在突变整合酶蛋白，这些核蛋白复合物中没有一个显示在野生型整合前复合物中检测到的天然整合体结构。我们的结论是，需要多种整合酶的功能，以形成感染细胞中的HIV-1整合体的天然结构。
Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200-250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wildtype preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells.