PDF(Pubmed)
Abstract：
HIV-1 integration into the human genome is dependent on 3\'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3\'-processed DNA remained resistant. Here, we describe the mechanism by which the 3\'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3\'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3\'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3\'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3\'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3\'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3\'-processed HIV-1 DNA.
摘要：
HIV-1整合到人类基因组中依赖于病毒DNA的3'-加工。最近,我们报道，细胞三总理修复外切酶1（TREX1）通过降解未加工的病毒DNA增强HIV-1整合，而整合胜任的3'处理的DNA仍然具有抗性。这里,我们描述了3'处理的HIV-1DNA抵抗TREX1介导的降解的机制。我们的动力学研究表明，通过TREX1，3'处理的DNA的切割速率（kcat）显着低于未处理的HIV-1DNA（约2-2.5倍）。处理过的U5和U3DNA底物的人TREX1的kcat值分别为3.8s-1和4.5s-1。相比之下,未处理的U5和U3基板分别在10.2s-1和9.8s-1下断裂。3'处理的DNA（U5-70.2和U3-28.05pM-1s-1）的降解效率（kcat/Km）也显着低于未处理的DNA（U5-103.1和U3-65.3pM-1s-1）。此外，与未加工的DNA相比，TREX1的结合亲和力(Kd)对3'处理的DNA显著较低(~2倍)。分子对接和动力学研究揭示了TREX1与3'处理和未处理的HIV-1DNA的不同构象结合模式。特别是,未加工的DNA有利地位于活性位点，与TREX1的催化残基发生极性相互作用。此外,与3'处理的DNA相比，TREX1和未加工的DNA之间形成了稳定的复合物。这些结果确定了TREX1优先降解整合能力不足的HIV-1DNA的机制，并揭示了整合能力3'处理的HIV-1DNA的独特结构和构象特性。
