Esposito D, Craigie R
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, 5 Center Drive MSC0560, Bethesda, MD 20892, USA.
EMBO J. 1998 Oct 1;17(19):5832-43. doi: 10.1093/emboj/17.19.5832.
HIV-1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C-terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence-specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction.
在逆转录病毒整合的起始步骤中，HIV-1整合酶特异性识别并切割病毒末端DNA。病毒末端DNA结合特异性的蛋白质和DNA决定因素尚未明确鉴定。我们利用病毒末端LTR序列的突变分析、体外筛选最佳病毒末端序列以及特异性光交联，来鉴定整合酶中与LTR末端特定碱基相互作用的区域。结果突出了整合酶核心结构域的无序环，特别是残基Q148和Y143，在与病毒DNA末端的末端部分结合中的作用。此外，我们确定了LTR末端上游与整合酶C末端结构域相互作用的位置，为该结构域在稳定病毒DNA结合中的作用提供了证据。最后，我们定位了一个以病毒DNA末端12个碱基为中心的区域，该区域在镁存在的情况下对病毒末端DNA结合似乎至关重要，但在锰存在的情况下并非如此，这表明二价阳离子对序列特异性结合有不同的影响。这些结果有助于确定整合酶与病毒DNA之间重要的接触区域，并有助于构建这种重要相互作用的分子模型。
