AN ESSENTIAL INTERACTION BETWEEN DISTINCT DOMAINS OF HIV-1 INTEGRASE MEDIATES ASSEMBLY OF THE ACTIVE MULTIMER
AN ESSENTIAL INTERACTION BETWEEN DISTINCT DOMAINS OF HIV-1 INTEGRASE MEDIATES ASSEMBLY OF THE ACTIVE MULTIMER
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DOI：
10.1074/jbc.270.7.3320
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发表时间：
1995-02-17
影响因子：
4.8
通讯作者：
BROWN, PO
中科院分区：
文献类型：
--
作者：
ELLISON, V;GERTON, J;BROWN, PO
整合酶介导逆转录病毒基因组整合到宿主细胞染色体中，这是病毒生命周期中的重要步骤。在体外，仅含有纯化的人免疫缺陷病毒（HIV）整合酶和模型病毒DNA底物前体的稳定复合物在整合反应中执行S '末端加工和DNA连接步骤。我们研究了HIV整合酶的三个基本组成部分之间的关系：HHCC结构域，一个假定的锌指，靠近N末端;遗传学保守的“DD 35 E”基序，它定义了催化结构域;以及一个通过其对烷化剂N-乙基马来酰亚胺（NEM）的敏感性而识别的特征。HIV整合酶是多聚体，并且这三种组分可以分布在多聚体酶的至少两个亚基中。这些组分在活性多聚体中不对称地起作用; DD 35 E基序和NEM敏感位点需要反式到HHCC区域。一个二价阳离子依赖性的相互作用，涉及一个整合酶亚基的NEM敏感位点和另一个亚基的HHCC区域点的作用，这两个功能的整合酶在多聚体组装。缺失HHCC结构域或用NEM修饰整合酶，损害了整合酶和病毒DNA之间稳定复合物的组装，表明整合途径中的这一初始步骤需要组装活性整合酶多聚体。
Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the S'-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved ''DD35E'' motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.