Identification of Site-Specific Adaptations Conferring Increased Neural Cell Tropism during Human Enterovirus 71 Infection

Cordey, Samuel; Petty, Tom J.; Schibler, Manuel; Martinez, Yannick; Gerlach, Daniel; Belle, Sandra Van; Turin, Lara; Zdobnov, Evgeny M.; Kaiser, Laurent; Tapparel, Caroline

doi:10.1371/journal.ppat.1002826

Cited by 91 publications

(106 citation statements)

References 73 publications

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“…A replacement of Glu with Lys at position 167 increased the positive electrostatic potential, which may restore their interactions between VP1 monomers, thereby stabilizing the capsid assembly. Interestingly, mutation E167G or E167A was found to be frequently associated with the VP1 L97R mutation, which conferred increased neural cell tropism during human EV71 infection (24), suggesting that residue E167 plays an central role in capsid assembly through interaction with residues L97 and R250. For mutant K274A, both mutations I111L and A224V could rescue the lethal phenotype of the K274A mutation.…”

Section: Discussionmentioning

confidence: 99%

“…Several key residues in the VP1 protein involved in pathogenesis have been identified. 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).…”

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confidence: 99%

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

Yuan

Wang

et al. 2016

J Virol

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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...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Yuan

Wang

et al. 2016

J Virol

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“…In a complex environment, the ability to generate a quasispecies may allow virus populations to respond and adapt fast (43)(44)(45)(46). Fidelity variants with wild-type-like replication kinetics are valuable tools to understand the roles of genetic diversity and mutation frequency in viral fitness and to evaluate the antiviral treatments (47).…”

Section: Discussionmentioning

confidence: 99%

Attenuation of Human Enterovirus 71 High-Replication-Fidelity Variants in AG129 Mice

Meng

Kwang

2014

J Virol

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In a screen for ribavirin resistance, a novel high-fidelity variant of human enterovirus 71 (EV71) with the single amino acid change L123F in its RNA-dependent RNA polymerase (RdRp or 3D) was identified. Based on the crystal structure of EV71 RdRp, L123 locates at the entrance of the RNA template binding channel, which might form a fidelity checkpoint. EV71 RdRp-L123F variants generated less progeny in a guanidine resistance assay and virus populations with lower mutation frequencies in cell culture passage due to their higher replication fidelity. However, compared with wild-type viruses, they did not show growth defects. In vivo infections further revealed that high-fidelity mutations L123F and G64R (previously reported) negatively impacted EV71 fitness and greatly reduced viral pathogenicity alone or together in AG129 mice. Interestingly, a variant with double mutations, RG/B4-G64R/L123F (where RG/B4 is an EV71 genotype B4 virus constructed by reverse genetics [RG])showed higher fidelity in vitro and less virulence in vivo than any one of the above two single mutants. The 50% lethal dose (LD 50 ) of the double mutant increased more than 500 times compared with the LD 50 of wild-type RG/B4 in mice. The results indicated that these high-fidelity variants exhibited an attenuated pathogenic phenotype in vivo and offer promise as a live attenuated EV71 vaccine. IMPORTANCEThe error-prone nature of the RNA-dependent RNA polymerase (RdRp) of RNA viruses during replication results in quasispecies and aids survival of virus populations under a wide range of selective pressures. Virus variants with higher replication fidelity exhibit lower genetic diversity and attenuated pathogenicity in vivo. Here, we identified a novel high-fidelity mutation L123F in the RdRp of human enterovirus 71 (EV71). We further elucidated that EV71 variants with the RdRp-L123F mutation and/or the previously identified high-fidelity mutation RdRp-G64R were attenuated in an AG129 mouse model. As EV71 has emerged as a serious worldwide health threat, especially in developing countries in the Asia-Pacific region, we urgently need EV71 vaccines. Learning from the poliovirus vaccination, we prefer live attenuated EV71 vaccines to inactivated EV71 vaccines in order to effectively control EV71 outbreaks at low cost. Our results imply a new means of attenuating EV71 and reducing its mutation rate at the same time.

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“…Analysis of whole genome sequences has been used to reveal epidemiologic and evolutionary dynamics of EVs through time and space [1,39,51]. Studies of EVs suggested that sequence variations in the UTRs, the VP1 region and the 3D polymerase could affect virus infection and replication capability in vitro and in vivo [103,104].…”

Section: Molecular Diversity and Evolution Of Enterovirusesmentioning

confidence: 99%

Molecular Analysis of Enteroviruses

Zhou

Sullivan

Iredell

et al. 2016

JHVRV

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Identification of Site-Specific Adaptations Conferring Increased Neural Cell Tropism during Human Enterovirus 71 Infection

Cited by 91 publications

References 73 publications

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

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

Attenuation of Human Enterovirus 71 High-Replication-Fidelity Variants in AG129 Mice

Molecular Analysis of Enteroviruses

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