Structural and molecular basis for Cardiovirus 2A as a viral gene switch

Programmed ribosome frameshifting in cardioviruses is activated by the 2A protein: a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here, we show that 2A binds directly to the frameshift-stimulatory element in the viral RNA with nanomolar affinity and equimolar stoichiometry, and we define the minimal RNA element required for binding. Through site-directed mutagenesis and the use of single-molecule optical tweezers, we study the dynamics of EMCV RNA (wild type- CCC as well CUC mutant), both alone and in the presence of 2A. By observing short-lived intermediate states in real-time, we demonstrate that the EMCV stimulatory element exists in at least two conformations and binding of 2A stabilizes one of these, an RNA pseudoknot, increasing the force required to unwind it.

在心病毒中，由2A蛋白触发的程序化核糖体移码现象被激活：这是一种多功能的致病因子，同时还能抑制帽依赖性翻译启动。本研究揭示，2A蛋白以纳摩尔亲和力和等摩尔化学计量直接结合到病毒RNA中的移码刺激元件，并确定了绑定所需的最低限度的RNA元件。通过位点特异性突变和单分子光镊技术，我们研究了埃姆斯病毒（EMCV）RNA（野生型-CCC以及CUC突变体）的动态特性，无论是单独存在还是与2A蛋白共存。通过实时观察短暂的中间状态，我们证实EMCV的刺激元件至少存在两种构象，2A蛋白的结合稳定了其中之一，即RNA假结，从而增加了解开该结构的所需力量。