Department of Structural Biology, University of Pittsburgh School of Medicine, Room 1037, Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
Department of Medicine, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Structure. 2019 Oct 1;27(10):1581-1593.e3. doi: 10.1016/j.str.2019.08.004. Epub 2019 Aug 27.
HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein that is proteolytically processed by HIV-1 protease (PR) to finally become a mature heterodimer, composed of a p66 and a p66-derived 51-kDa subunit, p51. Our previous work suggested that tRNA binding to p66/p66 introduces conformational changes in the ribonuclease (RNH) domain of RT that facilitate efficient cleavage of p66 to p51 by PR. In this study, we characterized the conformational changes in the RNH domain of p66/p66 imparted by tRNA using NMR. Moreover, the importance of tRNA in RT maturation was confirmed in cellulo by modulating the levels of Lys-tRNA synthetase, which affects recruitment of tRNA to the virus. We also employed nonnucleoside RT inhibitors, to modulate the p66 dimer-monomer equilibrium and monitor the resulting structural changes. Taken together, our data provide unique insights into the conformational changes in p66/p66 that drive PR cleavage.
HIV-1 逆转录酶（RT）作为 Gag-Pol 多聚蛋白的一部分被翻译，该多聚蛋白被 HIV-1 蛋白酶（PR）进行蛋白水解加工，最终成为成熟的异二聚体，由 p66 和 p66 衍生的 51kDa 亚基 p51 组成。我们之前的工作表明，tRNA 与 p66/p66 的结合会引起 RT 的核糖核酸酶（RNH）结构域发生构象变化，从而促进 PR 对 p66 进行有效的切割，形成 p51。在这项研究中，我们使用 NMR 技术对 p66/p66 中由 tRNA 引起的 RNH 结构域构象变化进行了表征。此外，通过调节赖氨酸 tRNA 合成酶的水平来改变 tRNA 对病毒的募集，从而在细胞内证实了 tRNA 在 RT 成熟过程中的重要性。我们还使用了非核苷类 RT 抑制剂来调节 p66 二聚体-单体平衡，并监测由此产生的结构变化。总之，我们的数据提供了对推动 PR 切割的 p66/p66 构象变化的独特见解。
