Chamond Nathalie, de Bisschop Grégoire, Faria Lisa, Laoudi Yamina, Martynov Valerii, Sargueil Bruno
Université Paris Cité, CNRS, CiTCoM Unit, PARIS F-75006, France.
Nucleic Acids Res. 2025 Aug 27;53(16). doi: 10.1093/nar/gkaf834.
DEAD-Box helicases are enzymes that bind and remodel RNA and ribonucleoproteins. They are involved in almost every step of RNA metabolism. DEAD-Box helicases are thus major players of gene expression (dys)-regulation and intracellular parasite invasion such as retroviruses. Among many implications in pathologies, the human DEAD-Box helicase DDX3X is hijacked by HIV-1 at various steps including viral RNA export from the nucleus and translation initiation, but little is known about the way it interacts with the viral RNA as well as the structural consequences of this interaction. Here, we show that DDX3X binds to specific regions of HIV-1 5'UTR and dissociates tightly bound dimers of HIV-1 RNA. Such enzymatic activity resulting in the destabilization of a complex structure in multiple turn-over conditions has never been observed with a DEAD-box helicase. DDX3X-induced dynamics was followed using time-resolved structure probing, while footprinting revealed DDX3X preferential binding sites. By coupling the biochemical analysis of DDX3X enzymatic activity the systematic probing of HIV-1-derived RNAs structure, we challenge both the accepted structural model of HIV-1 genomic RNA dimers as well as the dogma considering DEAD box proteins as inefficient and rather promiscuous towards their RNA substrates. An explicative mechanistic model is proposed.
DEAD盒解旋酶是一类能够结合并重塑RNA和核糖核蛋白的酶。它们几乎参与了RNA代谢的每一个步骤。因此，DEAD盒解旋酶是基因表达（失调）调控以及细胞内寄生虫入侵（如逆转录病毒）的主要参与者。在众多与疾病相关的影响中，人类DEAD盒解旋酶DDX3X在包括病毒RNA从细胞核输出和翻译起始在内的多个步骤中被HIV-1劫持，但人们对其与病毒RNA相互作用的方式以及这种相互作用的结构后果知之甚少。在这里，我们表明DDX3X与HIV-1 5'UTR的特定区域结合，并使紧密结合的HIV-1 RNA二聚体解离。在多轮反应条件下，这种导致复杂结构不稳定的酶活性从未在DEAD盒解旋酶中被观察到。使用时间分辨结构探测跟踪DDX3X诱导的动力学变化，而足迹分析揭示了DDX3X的优先结合位点。通过将DDX3X酶活性的生化分析与对HIV-1衍生RNA结构的系统探测相结合，我们对公认的HIV-1基因组RNA二聚体结构模型以及认为DEAD盒蛋白对其RNA底物效率低下且相当混杂的教条提出了挑战。我们提出了一个解释性的机制模型。
