Laboratory for Molecular Virology and Gene Therapy, Molecular Medicine, KULeuven and IRC Kulak, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium.
J Mol Biol. 2011 Jul 29;410(5):811-30. doi: 10.1016/j.jmb.2011.03.073.
Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.
虽然 LEDGF/p75 被认为通过将整合酶 (IN) 连接到染色质上来充当慢病毒整合的细胞辅助因子，但目前还没有很好的体外模型来分析这种功能。我们设计了一种 AlphaScreen 测定法来研究 LEDGF/p75 如何调节人类免疫缺陷病毒 1 型 IN 与 DNA 的相互作用。IN 与模拟长末端重复序列或随机 DNA 的 DNA 结合具有相似的亲和力。虽然 LEDGF/p75 与 DNA 结合紧密，但核定位信号 (NLS)/AT 钩功能受损的 LEDGF/p75 突变体与 DNA 结合较弱，而 LEDGF/p75 PWWP 结构域则没有相互作用，这与 NLS 和 AT 钩在电荷依赖性 DNA 结合中的作用的先前报道一致。LEDGF/p75 将 IN 与 DNA 的结合刺激了 10 倍至 30 倍。IN-DNA 结合的刺激需要 IN 和 LEDGF/p75 的 C 末端之间的直接相互作用。添加 LEDGF/p75 的 C 末端（氨基酸 325-530）或在 NLS/AT 钩中突变的 LEDGF/p75 会干扰 IN 与 DNA 的结合。我们的结果与 LEDGF/p75 介导的 IN 与 DNA 连接的体外模型一致。LEDGF/p75 C 末端对 IN-DNA 相互作用的抑制可能为抑制 HIV IN 活性提供了一种新策略，并可能解释了在细胞培养中过度表达 C 末端片段后观察到的 HIV 复制的强烈抑制。
