Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 1 avenue de la Terrasse, 91190, Gif-sur-Yvette, France.
BMC Biochem. 2018 Mar 21;19(1):2. doi: 10.1186/s12858-018-0092-x.
An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein.
In this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA.
These data support the conclusion that the complex formed between GagPol, mLysRS and tRNA, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA into HIV-1 particles.
人类免疫缺陷病毒 1 型（HIV-1）复制的一个重要步骤是包装来自宿主细胞的 tRNA，该 tRNA 在逆转录起始过程中充当引物 RNA。病毒 GagPol 多蛋白前体和来自宿主细胞的人线粒体赖氨酰-tRNA 合成酶（mLysRS）被认为参与了包装过程。更具体地说，mLysRS 的催化结构域被认为与 GagPol 多蛋白的 Pol 区的反式框架（TF 或 p6*）和整合酶（IN）结构域相互作用。
在这项工作中，我们报告了 mLysRS 与其病毒伙伴（Pol 多蛋白和分离的整合酶和反式框架域）之间的蛋白质-蛋白质相互作用的定量特征。确定了 Pol:mLysRS 相互作用的离解常数为 1.3±0.2 nM，这证明了这种相互作用的稳健性。GagPol 的蛋白酶和逆转录酶结构域在这种相互作用中是可有可无的，但 TF 和 IN 结构域必须通过连接多肽连接，以重现 mLysRS 的高亲和力伴侣。病毒蛋白与 mLysRS 的结合并没有显著提高 mLysRS 对 tRNA 的结合亲和力。
这些数据支持以下结论：即 GagPol、mLysRS 和 tRNA 形成的复合物涉及 Pol 的 IN 和 TF 结构域与 mLysRS 的直接相互作用，比以前认为参与 HIV-1 颗粒中 tRNA 包装的模型所假设的复合物更为稳健。
