Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease

ABSTRACT Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (re...

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Autores principales:	Maria L. Agostini, Erica L. Andres, Amy C. Sims, Rachel L. Graham, Timothy P. Sheahan, Xiaotao Lu, Everett Clinton Smith, James Brett Case, Joy Y. Feng, Robert Jordan, Adrian S. Ray, Tomas Cihlar, Dustin Siegel, Richard L. Mackman, Michael O. Clarke, Ralph S. Baric, Mark R. Denison
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	RNA polymerases SARS-CoV antiviral agents antiviral resistance coronavirus nucleoside analogs Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1b011f9fd4ae445884489fb5ef613ee9
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spelling	oai:doaj.org-article:1b011f9fd4ae445884489fb5ef613ee92021-11-15T15:53:26ZCoronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease10.1128/mBio.00221-182150-7511https://doaj.org/article/1b011f9fd4ae445884489fb5ef613ee92018-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00221-18https://doaj.org/toc/2150-7511ABSTRACT Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50. The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs. IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.Maria L. AgostiniErica L. AndresAmy C. SimsRachel L. GrahamTimothy P. SheahanXiaotao LuEverett Clinton SmithJames Brett CaseJoy Y. FengRobert JordanAdrian S. RayTomas CihlarDustin SiegelRichard L. MackmanMichael O. ClarkeRalph S. BaricMark R. DenisonAmerican Society for MicrobiologyarticleRNA polymerasesSARS-CoVantiviral agentsantiviral resistancecoronavirusnucleoside analogsMicrobiologyQR1-502ENmBio, Vol 9, Iss 2 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	RNA polymerases SARS-CoV antiviral agents antiviral resistance coronavirus nucleoside analogs Microbiology QR1-502
spellingShingle	RNA polymerases SARS-CoV antiviral agents antiviral resistance coronavirus nucleoside analogs Microbiology QR1-502 Maria L. Agostini Erica L. Andres Amy C. Sims Rachel L. Graham Timothy P. Sheahan Xiaotao Lu Everett Clinton Smith James Brett Case Joy Y. Feng Robert Jordan Adrian S. Ray Tomas Cihlar Dustin Siegel Richard L. Mackman Michael O. Clarke Ralph S. Baric Mark R. Denison Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease
description	ABSTRACT Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50. The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs. IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.
format	article
author	Maria L. Agostini Erica L. Andres Amy C. Sims Rachel L. Graham Timothy P. Sheahan Xiaotao Lu Everett Clinton Smith James Brett Case Joy Y. Feng Robert Jordan Adrian S. Ray Tomas Cihlar Dustin Siegel Richard L. Mackman Michael O. Clarke Ralph S. Baric Mark R. Denison
author_facet	Maria L. Agostini Erica L. Andres Amy C. Sims Rachel L. Graham Timothy P. Sheahan Xiaotao Lu Everett Clinton Smith James Brett Case Joy Y. Feng Robert Jordan Adrian S. Ray Tomas Cihlar Dustin Siegel Richard L. Mackman Michael O. Clarke Ralph S. Baric Mark R. Denison
author_sort	Maria L. Agostini
title	Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease
title_short	Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease
title_full	Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease
title_fullStr	Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease
title_full_unstemmed	Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease
title_sort	coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/1b011f9fd4ae445884489fb5ef613ee9
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Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease

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