Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation
Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation
复制标题
肠道病毒 71 RNA 依赖性 RNA 聚合酶与其蛋白引物 VPg 复合的晶体结构：VPg 尿苷酰化反式机制的意义
DOI：
10.1128/jvi.02733-12
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发表时间：
2013-05-01
影响因子：
5.4
通讯作者：
Lou, Zhiyong
中科院分区：
文献类型：
--
作者：
Chen, Cheng;Wang, Yaxin;Lou, Zhiyong
摘要短小核糖核酸病毒的RNA复制是由VPG尿苷基化启动的，在此过程中，病毒编码蛋白VPG的第三个酪氨酸残基的羟基通过RNA依赖的RNA聚合酶(RdRp；也称为3Dpol)共价连接到两个UMP分子上。我们先前确定位于肠道病毒71(EV71)RdRp棕榈区碱基的311位是EV71 VPG结合和尿苷基化的位点。本文报道了与VPG络合的EV713DPoll的晶体结构。VPG锚定在3Dpol分子手掌结构域的底部，呈延伸的V型构象。3Dpol上的相应界面主要由311位残基和手掌和手指结构域中的其他残基组成。参与VPG相互作用的3Dpol氨基酸(3DL319A、3DD320A和3DY335A)的突变显著破坏了VPG与3Dpol的结合，导致VPG尿苷基化缺陷。相反，这些突变并不影响3Dpol的RNA延伸活性。在病毒基因组RNA的背景下，取消VPG尿苷基化活性的突变对EV71的复制是致命的。进一步的体外分析表明，VPG结合缺陷突变体和催化缺陷突变体混合后尿苷基化活性恢复，提示EV71 VPG尿苷基化的反式作用机制。我们的结果与其他研究的结果一起表明，不同的微小核糖核酸病毒对VPG尿苷基化使用不同的结合位点。
ABSTRACT Picornavirus RNA replication is initiated by VPg uridylylation, during which the hydroxyl group of the third tyrosine residue of the virally encoded protein VPg is covalently linked to two UMP molecules by RNA-dependent RNA polymerase (RdRp; also known as 3Dpol). We previously identified site 311, located at the base of the palm domain of the enterovirus 71 (EV71) RdRp, to be the site for EV71 VPg binding and uridylylation. Here we report the crystal structure of EV71 3Dpol complexed with VPg. VPg was anchored at the bottom of the palm domain of the 3Dpol molecule and exhibited an extended V-shape conformation. The corresponding interface on 3Dpol was mainly formed by residues within site 311 and other residues in the palm and finger domains. Mutations of the amino acids of 3Dpol involved in the VPg interaction (3DL319A, 3DD320A, and 3DY335A) significantly disrupted VPg binding to 3Dpol, resulting in defective VPg uridylylation. In contrast, these mutations did not affect the RNA elongation activity of 3Dpol. In the context of viral genomic RNA, mutations that abolished VPg uridylylation activity were lethal for EV71 replication. Further in vitro analysis showed that the uridylylation activity was restored by mixing VPg-binding-defective and catalysis-defective mutants, indicating a trans mechanism for EV71 VPg uridylylation. Our results, together with previous results of other studies, demonstrate that different picornaviruses use distinct binding sites for VPg uridylylation.