Selection and characterisation of guanidine-resistant mutants of human enterovirus 71

Sadeghipour, Sara; Bek, Emily J.; McMinn, Peter C.

doi:10.1016/j.virusres.2012.07.005

Cited by 25 publications

(27 citation statements)

References 44 publications

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“…Guanidine resistance in picornaviruses is associated with the development of point mutations in coding region 2C (16,21,23,24), including HEV71 (22). After 13 passages in RD cells in the presence of 400 M ribavirin, we showed that the G64R and G64T virus populations grew poorly in the presence of 0.5 mM guanidine relative to growth of the G64N and parental virus populations.…”

Section: Discussionmentioning

confidence: 82%

“…In the poliovirus model, the rate of random incorporation of guanidine resistance mutations, located in gene 2C (16,21), has been used to estimate the replication fidelity of ribavirinresistant mutants (10,13). We recently showed that resistance to guanidine in HEV71 is associated with several amino acid substitutions in coding region 2C (22). Thus, we developed a guanidine resistance assay to assess the replication fidelity of the parental virus and the G64N, G64R, and G64T mutant virus populations.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, no mutations were found in the 2C coding regions of any of the plaque-purified G64R and G64T mutant populations at RD passage 1 or 13 ( Table 2). It is interesting to note that the M175K, K185R, E272K, R293K, and K298N mutations in coding region 2C have not been shown to confer guanidine resistance upon HEV71 (1) or upon other picornaviruses (16,(21)(22)(23)(24).…”

Section: Hev71 3d Mutants With High Replication Fidelitymentioning

confidence: 99%

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Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

Sadeghipour

Bek

McMinn

2013

J Virol

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It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 M ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 M ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 M ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.

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Section: Discussionmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Hev71 3d Mutants With High Replication Fidelitymentioning

confidence: 99%

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Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

Sadeghipour

Bek

McMinn

2013

J Virol

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“…However, the crystal structure of EV71 RdRp reveals that the location of L123 excludes the possible interaction between L123 and ribavirin (Fig. 4), and, more, the RdRp-L123F variants resisted another drug as well, guanidine, which directly targets 2C but not RdRp (38). Therefore, we prefer the third mechanism, i. e., that RdRp-L123F confers a general increase in RNA replication fidelity.…”

Section: Discussionmentioning

confidence: 99%

“…To test the possibility that the ribavirin resistance of L123F variants results from the increased replication fidelity of RdRp, as previously reported in poliovirus (8), a guanidine resistance assay and mutation frequency test of RG/B4 and RG/B4-L123F were performed. EV71 resistance to guanidine can be generated by several amino acid substitutions in the 2C coding region (38). From the rate of random incorporation of guanidine resistance mutations in 2C, the replication fidelity of RG/B4 and RG/B4-L123F was estimated.…”

Section: Generation Of Ribavirin-resistant Ev71-b5 Variantsmentioning

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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Combating coxsackievirus B infections

Nekoua

Mercier

Vergez

et al. 2022

Reviews in Medical Virology

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Coxsackieviruses B (CVB) are small, non‐enveloped, single‐stranded RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. They are common worldwide and cause a wide variety of human diseases ranging from those having relatively mild symptoms to severe acute and chronic pathologies such as cardiomyopathy and type 1 diabetes. The development of safe and effective strategies to combat these viruses remains a challenge. The present review outlines current approaches to control CVB infections and associated diseases. Various drugs targeting viral or host proteins involved in viral replication as well as vaccines have been developed and shown potential to prevent or combat CVB infections in vitro and in vivo in animal models. Repurposed drugs and alternative strategies targeting miRNAs or based on plant extracts and probiotics and their derivatives have also shown antiviral effects against CVB. In addition, clinical trials with vaccines and drugs are underway and offer hope for the prevention or treatment of CVB‐induced diseases.

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Selection and characterisation of guanidine-resistant mutants of human enterovirus 71

Cited by 25 publications

References 44 publications

Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

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

Combating coxsackievirus B infections

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