School of Life Sciences, University of Science and Technology of China, Hefei, China.
Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China.
J Virol. 2024 Oct 22;98(10):e0091124. doi: 10.1128/jvi.00911-24. Epub 2024 Sep 6.
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.
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.
2C 是一种高度保守的小核糖核酸病毒非结构蛋白，具有 ATP 酶活性，在病毒生命周期中发挥多种功能，是抗小核糖核酸病毒药物开发的有希望的靶点。虽然肠病毒 2C 的结构-功能已经得到了很好的研究，但心脏病毒 2C 仍然很大程度上没有被描述。在这里，从脑炎心肌炎病毒（肠病毒 A）的晶体结构中鉴定出一种内源性 ATP 分子。ATP 结合到 ATP 酶活性部位，具有独特的紧凑构象。值得注意的是，ATP 的γ-磷酸与精氨酸 311（心脏病毒 2C 中保守）直接相互作用，其突变显著抑制 ATP 酶活性。出乎意料的是，这种突变显著促进 2C 自身寡聚化和病毒复制效率。分子动力学模拟表明，精氨酸 311 侧链高度动态，表明它可能在 ATP 酶活性的激活状态和抑制状态之间发挥开关作用。脑炎心肌炎病毒 2C 全长六聚体环模型表明，C 端螺旋可能靠近 N 端两亲性螺旋，形成一个连续的正区域用于 RNA 结合。脑炎心肌炎病毒 2C 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，以指导脑炎心肌炎病毒 2C 的表征和精氨酸在心脏病毒 2C 功能中的重要作用。
脑炎心肌炎病毒（肠病毒 A）是同名疾病的病原体，可在各种哺乳动物中引起心肌炎、脑炎和生殖障碍。2C 蛋白在功能上是不可或缺的，是广谱小核糖核酸病毒抑制剂开发的有希望的靶点。在这里，通过蛋白质晶体学和高效液相色谱的组合，在脑炎心肌炎病毒（EMCV）2C 结构中发现了一种具有独特构象的内源性 ATP 分子。脑炎心肌炎病毒 2C 的生化和结构特征分析揭示了保守的精氨酸 311 在 ATP 酶活性和脑炎心肌炎病毒 2C 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，该残基负调控脑炎心肌炎病毒的感染滴度和病毒包封效率，因此对 2C 蛋白促进病毒复制至关重要。我们系统的结构-功能分析为心脏病毒 2C 蛋白的功能和调节机制提供了独特的见解。
