Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain.
J Virol. 2014 May;88(10):5595-607. doi: 10.1128/JVI.03502-13. Epub 2014 Mar 5.
Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A.
The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.
心肌炎病毒（EMCV）是小核糖核酸病毒科中冠状病毒属的一个成员，该科包括许多重要的人类和动物病原体。RNA 依赖性 RNA 聚合酶（RdRp）3Dpol 是病毒基因组复制的关键酶。在这项研究中，我们报告了两种不同晶体形式的 EMCV RdRp 的 X 射线结构，其分辨率分别为 2.8 和 2.15Å。还证明了纯化酶的体外延伸和 VPg 尿苷酸化活性。尽管 EMCV 3Dpol 的整体结构与已知的 Picornaviridae 家族其他成员的 RdRps 相似，但结构比较表明，其中一种晶体形式的活性部位腔发生了很大的重组。这种重排主要影响 A 基序，其中参与核糖核苷三磷酸（rNTP）选择的保守残基天冬氨酸 240 和其相邻残基苯丙氨酸 239，从其在核糖结合口袋内的预期位置移动约 10Å，朝向 rNTP 隧道的入口。A 基序的这种改变构象通过苯丙氨酸 239 和手指结构域内赖氨酸 56 之间建立的阳离子-π 相互作用得到稳定。还观察到涉及苯丙氨酸 239 和 F 基序的不同残基的其他接触。A 基序的运动与残基 54 到 63 之间侧翼的手指区域、包含赖氨酸 56 的聚合酶 N 端以及侧翼的重要构象变化相关。这项工作中确定的结构为研究心血管病毒 RNA 复制过程提供了重要信息，并且可能对开发针对 A 基序改变构象的新型抗病毒药物具有重要意义。
小核糖核酸病毒科是已知最大的病毒科之一，包括许多重要的人类和动物病原体。RNA 依赖性 RNA 聚合酶（RdRp）3Dpol 是小核糖核酸病毒基因组复制的关键酶，也是开发抗病毒疗法的有效靶点。通过解析第一个心血管病毒 RdRp（EMCV 3Dpol）的 X 射线结构，我们捕获了聚合酶活性部位中保守基序（A 基序）的改变构象，该基序包含参与 rNTP 选择和结合的天冬氨酸残基。这种 A 基序的改变构象通过小核糖核酸病毒中严格保守的许多残基稳定，干扰 rNTP 底物在活性部位中的正确定位。观察到的重排表明，该 A 基序片段是一个动态元件，可以通过外部效应物进行调节，无论是激活还是抑制酶活性，并且这种类型的调节似乎对所有小核糖核酸病毒都是通用的。
