2C是一种高度保守的小核糖核酸病毒非结构蛋白,具有ATPase活性,在病毒生命周期中起着多功能作用,是抗小核糖核酸病毒药物开发的有希望的靶标。虽然肠道病毒2Cs的结构功能已经得到了很好的研究,心病毒2Cs在很大程度上仍未表征。这里,在来自脑心肌炎病毒的2C的晶体结构中鉴定出内源性ATP分子(EMCV,心病毒A)。ATP以独特的紧密构象结合到ATP酶活性位点中。值得注意的是,ATP的γ-磷酸直接与Arg311相互作用(在心病毒2Cs中保守),其突变显著抑制ATP酶活性。出乎意料的是,该突变显著促进2C自身寡聚化和病毒复制效率。分子动力学模拟表明,Arg311侧链是高度动态的,表明它可以作为ATPase活性的激活状态和抑制状态之间的转换。EMCV2C全长的六聚体环模型表明,C端螺旋可能接近N端两亲性螺旋,形成RNA结合的连续阳性区域。EMCV2C的RNA结合研究表明,RNA长度与RNA结合亲和力密切相关,并表明底物可能包裹在六聚体的外表面。我们的研究提供了一个生化框架来指导EMCV2C的表征和精氨酸在心血管病毒2C功能中的重要作用。
目的:脑心肌炎病毒(心脏病毒A)是同型疾病的病原体,这可能会诱发心肌炎,脑炎,和各种哺乳动物的生殖障碍。2C蛋白在功能上是必不可少的,并且是涉及广谱小核糖核酸病毒抑制剂的药物开发的有希望的靶标。这里,通过蛋白质晶体学和高效液相色谱法在脑心肌炎病毒(EMCV)2C结构中发现了具有独特构象的内源性ATP分子.EMCV2C的生化和结构表征分析揭示了保守的Arg311在ATPase活性和EMCV2C的自身寡聚化中的关键作用。病毒复制动力学和感染性研究表明,该残基对EMCV的感染性滴度和病毒包封率具有负调控作用,因此,2C蛋白对促进病毒复制至关重要。我们的系统结构功能分析为心血管病毒2C蛋白的功能和调节机制提供了独特的见解。
2C is a highly conserved picornaviral non-structural protein with ATPase activity and plays a multifunctional role in the viral life cycle as a promising target for anti-picornavirus drug development. While the structure-function of enteroviral 2Cs have been well studied, cardioviral 2Cs remain largely uncharacterized. Here, an endogenous ATP molecule was identified in the crystal structure of 2C from encephalomyocarditis virus (EMCV, Cardiovirus A). The ATP is bound into the ATPase active site with a unique compact conformation. Notably, the γ-phosphate of ATP directly interacts with Arg311 (conserved in cardioviral 2Cs), and its mutation significantly inhibits the ATPase activity. Unexpectedly, this mutation remarkably promotes 2C self-oligomerization and viral replication efficiency. Molecular dynamic simulations showed that the Arg311 side chain is highly dynamic, indicating it may function as a switch between the activation state and the inhibition state of ATPase activity. A hexameric ring model of EMCV 2C full length indicated that the C-terminal helix may get close to the N-terminal amphipathic helices to form a continuous positive region for RNA binding. The RNA-binding studies of EMCV 2C revealed that the RNA length is closely associated with the RNA-binding affinities and indicated that the substrate may wrap around the outer surface of the hexamer. Our studies provide a biochemical framework to guide the characterization of EMCV 2C and the essential role of arginine in cardioviral 2C functions.
OBJECTIVE: Encephalomyocarditis virus (Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis, and reproductive disorders in various mammals. 2C protein is functionally indispensable and a promising target for drug development involving broad-spectrum picornaviral inhibitors. Here, an endogenous ATP molecule with a unique conformation was discovered by a combination of protein crystallography and high-performance liquid chromatography in the encephalomyocarditis virus (EMCV) 2C structure. Biochemical and structural characterization analysis of EMCV 2C revealed the critical role of conserved Arg311 in ATPase activity and self-oligomerization of EMCV 2C. The viral replication kinetics and infectivity study suggested that the residue negatively regulated the infectivity titer and virus encapsulation efficiency of EMCV and is, therefore, crucial for 2C protein to promote viral replication. Our systemic structure-function analysis provides unique insights into the function and regulation mechanism of cardioviral 2C protein.