Ciuffi Angela, Diamond Tracy L, Hwang Young, Marshall Heather M, Bushman Frederic D
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6076, USA.
Hum Gene Ther. 2006 Sep;17(9):960-7. doi: 10.1089/hum.2006.17.960.
The mechanisms controlling retroviral integration have been the topic of intense interest, in part because of adverse clinical events that occurred during retrovirus-mediated human gene therapy. Here we investigate the use of artificial tethering interactions to constrain retroviral integration site selection in an in vitro model. During normal infection, HIV DNA integration is favored in active cellular transcription units. One component of the targeting mechanism is the cellular LEDGF/p75 protein. LEDGF/p75 binds tightly to HIV integrase (IN) protein, and depletion of LEDGF/p75 from target cells results in reduced integration in transcription units, suggesting integration targeting by a tethering mechanism. We constructed and analyzed fusions of LEDGF/p75 or its IN-binding domain (IBD) to the DNA-binding domain of phage lambda repressor protein (lambdaR). In the presence of the lambdaR-LEDGF/p75 fusions, increased strand transfer by IN was seen in target DNA near lambdaR-binding sites in vitro . These data support the idea that a direct interaction between LEDGF/p75 and IN can mediate targeting via a tethering mechanism, and provide proof of concept for the idea that protein-protein interactions might be engineered to constrain integration site selection during human gene therapy.
控制逆转录病毒整合的机制一直是人们高度关注的话题，部分原因是逆转录病毒介导的人类基因治疗期间发生了不良临床事件。在此，我们在体外模型中研究利用人工系留相互作用来限制逆转录病毒整合位点的选择。在正常感染过程中，HIV DNA整合在活跃的细胞转录单元中更受青睐。靶向机制的一个组成部分是细胞LEDGF/p75蛋白。LEDGF/p75与HIV整合酶（IN）蛋白紧密结合，从靶细胞中耗尽LEDGF/p75会导致转录单元中的整合减少，这表明通过系留机制进行整合靶向。我们构建并分析了LEDGF/p75或其IN结合结构域（IBD）与噬菌体λ阻遏蛋白（λR）的DNA结合结构域的融合体。在存在λR-LEDGF/p75融合体的情况下，体外在靠近λR结合位点的靶DNA中观察到IN介导的链转移增加。这些数据支持LEDGF/p75与IN之间的直接相互作用可通过系留机制介导靶向的观点，并为在人类基因治疗期间可设计蛋白质-蛋白质相互作用来限制整合位点选择的观点提供了概念验证。
