Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation

Abstract

g 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 3D pol ). 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 3D pol complexed with VPg. VPg was anchored at the bottom of the palm domain of the 3D pol molecule and exhibited an extended V-shape conformation. The corresponding interface on 3D pol was mainly formed by residues within site 311 and other residues in the palm and finger domains. Mutations of the amino acids of 3D pol involved in the VPg interaction ( 3D L319A, 3D D320A, and 3D Y335A) significantly disrupted VPg binding to 3D pol , resulting in defective VPg uridylylation. In contrast, these mutations did not affect the RNA elongation activity of 3D pol . 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. Picornaviridae members make up one of the largest family of viruses and cause a wide range of infectious diseases in plants, animals, and humans. As the members of Picornaviridae, enterovirus 71 (EV71) and coxsackievirus (CV) are the major causative agents of hand, foot, and mouth disease (HFMD) in mainland China (1, 2). These agents caused over 1,000,000 infections and 900 deaths in 2010

Initiation of protein-primed picornavirus RNA synthesis

“…One possible explanation of how VPg could be uridylylated at this site is that the nucleotidylylation reaction is carried out by a second molecule of 3D pol (59, 103, 151). Such a “trans” uridylylation mechanism was supported also from structural studies of the EV71 polymerase complexed with VPg, in which VPg bound at the bottom of the palm domain could not access the catalytic site (27). It should be noted, however, that a front-loading model of VPg-binding and a “ cis ” uridylylation mechanism was proposed for FMDV 3D pol (43) and for the RNA polymerase of rhinovirus (7).…”

“…Three dimensional structures have been reported for several members of the virus family such as PV, HRV14, CVB3, FMDV, EV-71 (7, 27, 43, 44, 59, 61). The 3D pol of Picornaviridae are 460-470 amino acids long and possess high primary sequence similarity and structural homology (26).…”

Peptidyl Aldehyde NK-1.8k Suppresses Enterovirus 71 and Enterovirus 68 Infection by Targeting Protease 3C

“…The EV genome contains a single-stranded positive-sense polyadenylated RNA of approximately 7,400 nucleotides (10). Translation of the single open reading frame is initiated by ribosomes that use an internal ribosomal entry site located in the 5= untranslated region (UTR) region of the viral genome and gives rise to a polyprotein of approximately 250 kDa (11)(12)(13)(14). This polyprotein is further processed into four structural proteins (VP1 to VP4) to form the viral capsid and seven structural proteins (2A to 2C and 3A to 3D) by two viral proteases (2A and protease 3C [3C pro ]), together with a protease precursor, 3CD (15).…”

Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation

“…This suggests that the VPg bound at this site in 3D pol cannot be uridylylated by the carrier 3D pol , which raises the possibility of an intermolecular uridylylation reaction in which two molecules of 3D pol are required for VPgpUpU synthesis (Tellez et al, 2006;Gruez et al, 2008). In recent studies using EV-71, a similar model for intermolecular uridylylation has been proposed (Chen et al, 2013;Sun et al, 2012). In earlier studies, mutations in the VPg binding site on the back of PV1 3D pol were shown to inhibit 3AB binding to 3D pol (Hope et al, 1997;Lyle et al, 2002).…”