Genetic characterization of enterovirus 71 isolated from patients with severe disease by comparative analysis of complete genomes

bEnterovirus 71 (EV71) is an important emerging human pathogen with a global distribution and presents a disease pattern resembling poliomyelitis with seasonal epidemics that include cases of severe neurological complications, such as acute flaccid paralysis. EV71 is a member of the Picornaviridae family, which consists of icosahedral, nonenveloped, single-stranded RNA viruses. Here we report structures derived from X-ray crystallography and cryoelectron microscopy (cryo-EM) for the 1095 strain of EV71, including a putative precursor in virus assembly, the procapsid, and the mature virus capsid. The cryo-EM map of the procapsid provides new structural information on portions of the capsid proteins VP0 and VP1 that are disordered in the higher-resolution crystal structures. Our structures solved from virus particles in solution are largely in agreement with those from prior X-ray crystallographic studies; however, we observe small but significant structural differences for the 1095 procapsid compared to a structure solved in a previous study (

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Structures of the Procapsid and Mature Virion of Enterovirus 71 Strain 1095

Cifuente

Lee

Yoder

et al. 2013

“…6) (16, 22). VP1-145 has been found to be under positive selection (36,37) and has been implicated as one of the possible determinants of virulence in humans (38,39) and adaptation in the mouse (40)(41)(42). It is likely that the EV71 antigenicity, as well as receptor-binding phenotype, is altered with a single mutation at VP1-145, which may confer an advantage to EV71 for effective host infection and transmission.…”

Section: Discussionmentioning

confidence: 99%

A Strain-Specific Epitope of Enterovirus 71 Identified by Cryo-Electron Microscopy of the Complex with Fab from Neutralizing Antibody

Lee

Cifuente

Ashley

et al. 2013

e Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody. E nterovirus 71 (EV71) infection causes outbreaks of hand, foot, and mouth disease (HFMD), predominantly in the Asia-Pacific region (1, 2). Whereas most EV71 infections are self-limiting, neurological and systemic complications can develop that range from aseptic meningitis to encephalitis and acute flaccid paralysis. Infection can lead to lethal pulmonary edema and heart failure (2), with mortality being especially high in young children under the age of 6 (2, 3). As seasonal outbreaks of HFMD are recurring around the world, development of a vaccine and antiviral therapies for EV71 has become an urgent concern.A member of the Picornaviridae family, EV71 has a nonenveloped, icosahedral capsid comprised of 60 copies of each of four viral structural proteins (VP1 to VP4) (4). Recent studies have solved the structures for three strains of EV71 (MY104 [5], Fuyang [6], and 1095 [7]), demonstrating that EV71 has the general features of picornavirus capsids, including the 5-fold "mesa" and the depression around the mesa called the "canyon" (5-8). Conserved residues VP1-242K and VP1-244K form positively charged patches on the 5-fold mesa (6), and this symmetry-related clustering of positive charges has been suggested as a common mechanism for heparan sulfate binding in enteroviruses (9).Several cellular receptors for EV71 have been identified: scavenger receptor B2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), and heparan sulfate (HS) (10-12). SCARB2, which is expressed on a broad variety of cell types, likely binds to the virus canyon and induces the transition of the virion that is required for uncoating (13-15). PSGL-1, which is expressed exclusively on lymphocytes, binds only specific EV71 strains and supports viral replication in lymphocytes in a PSGL-1-dependent manner (11). According to recent studies, PSGL-1 and HS bind the positively charged patches on the 5-fold mesa of EV71 and provide initial attachment on the cell (12,16,17). We recently found that the PSGL-1 binding phenotype of EV71 strains is regulated b...

“…A nonconservative amino acid change in VP1 located within the BC loop (L97R) contributes to dissemination and neurotropism in immunocompromised patients (24). The residue at position 145 of VP1 (VP1-145) controls virus tropism by changing the accessibility of the positively charged lysine side chain of VP1-244 to the negatively charged N terminus of PSGL-1 on leukocytes (25) and has been implicated as one of the possible determinants of virulence in humans (26,27). Moreover, VP1 is an attractive target for identification of EV71 inhibitors.…”

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confidence: 99%

Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles

Yuan

Wang

et al. 2016

Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra-and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles. IMPORTANCEInfection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are currently available for the prevention or treatment of EV71 infection. Understanding the determinants of virion assembly and entry will facilitate vaccine development and drug discovery. Here, we identified 23 out of 27 positively charged residues in VP1 which impaired or blocked the production of infectious particles. The defect could be rescued by second-site mutations within the VP1 protein. Our findings highlight the importance of positively charged residues in VP1 during infectious particle production and reveal a potential strategy for blocking EV71 infections by inhibiting intra-or intermolecular interactions of the VP1 protein. Enterovirus 71 (EV71) is a nonenveloped icosahedral RNA virus belonging to genus Enterovirus within the family Picornaviridae. Since its first isolation from the stool of an infant with encephalitis in California in 1969 (1), EV71 has been recognized as a major cause of hand, foot, and mouth disease (HFMD), which is associated with severe neurological symptoms in a small proportion of cases (2). There has been a significant incre...