Abstract
ABSTRACTEmerging 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.
Highlights
Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available
We recently reported that GS-5734, the monophosphoramidate prodrug of the C-adenosine nucleoside analogue GS-441524 (Fig. 1A), inhibits SARS-CoV, MERS-CoV, and bat CoV strains that are capable of replicating in primary human airway epithelial cells and mediate entry using human CoV receptors [23–25]
We observed minimal detectable cytotoxicity within the tested range, with the concentration resulting in 50% cytotoxicity (CC50) Ͼ300 M (Fig. 1D)
Summary
Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The results indicate that GS-5734 interferes with the nsp 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. Identification and development of antiviral nucleosides against coronaviruses have been hampered by the presence of the unique CoV proofreading 3=-5= exoribonuclease (ExoN) [16–18] While nucleoside analogues such as BCX4430 inhibit CoVs [19], several previously tested nucleoside analogues have been incapable of potently inhibiting CoV replication, and others have demonstrated poor selectivity indexes [20, 21]. We have shown that CoV resistance to the mutagens 5-fluorouracil (5-FU) and ribavirin (RBV) in vitro is attributed to their removal by the proofreading ExoN [22], supporting the hypothesis that an effective nucleoside analogue must evade proofreading to successfully interfere with CoV RNA synthesis
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