Crystal structure of enterovirus 71 RNA-dependent RNA polymerase complexed with its protein primer VPg: Implication for a trans mechanism of VPg uridylylation

Cheng Chen, Yaxin Wang, Chao Shan, Yuna Sun, Peng Xu, Honggang Zhou, Cheng Yang, Pei Yong Shi, Zihe Rao, Bo Zhang, Zhiyong Lou

Research output: Contribution to journalArticle

42 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)5755-5768
Number of pages14
JournalJournal of virology
Volume87
Issue number10
DOIs
StatePublished - May 1 2013

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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