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 (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.

Arguments in favour of remdesivir for treating SARS-CoV-2 infections

“…The chemical formula of remdesivir with a molecular mass of 602.6 is C 27 H 35 N 6 O 8 P. In several human cell lines, remdesivir can be effectively metabolized to active nucleoside triphosphate [15]. An in vitro study has demonstrated that nucleoside triphosphate works as an incorporation competitor with adenosine triphosphate, confuses viral RdRp, acts as a delayed RNA-chain terminator against Ebola virus [15,16], evades proofreading by viral exoribonuclease, and causes a decrease in viral RNA production [17]. Recently, the antiviral activity of remdesivir was exhibited at the stage after virus entry into Vero E6 cells, supporting its antiviral mechanism as a nucleotide analog [18].…”

“…Even before approval and clinical use, the concern of antiviral resistance in remdesivir has been studied. Using MHV as the tested CoV, two of three lineages of wild-type MHV in the presence of increased concentrations of GS-441524 were lost after 17 and 20 repeated passages, and only one lineage after 23 passages selected a low-level resistant mutant, conferring a 5.6-fold increase in EC 50 [17]. Two amino acid substitutions (F467L and V553L) were noted in non-structural protein (nsp) 12, the RdRp of MHV, and also resulted in a 6-fold increase of EC 50 in SARS-CoV.…”

Advances in respiratory virus therapeutics – A meeting report from the 6th isirv Antiviral Group conference

“…In parallel, in vitro passage studies were also performed to determine if coronavirus genomes could evolve to replicate in the presence of remdesivir. After 23 passages in the presence of drug, two mutations were identified in the viral RNA dependent RNA polymerase gene (F276L and V553L) that allowed for increased replication in the presence of remdesivir (Agostini et al, 2018). These two amino acid changes decrease viral fitness overall and attenuate SARS-CoV pathogenesis in a mouse model of SARS-CoV induced disease (Agostini et al, 2018).…”

“…After 23 passages in the presence of drug, two mutations were identified in the viral RNA dependent RNA polymerase gene (F276L and V553L) that allowed for increased replication in the presence of remdesivir (Agostini et al, 2018). These two amino acid changes decrease viral fitness overall and attenuate SARS-CoV pathogenesis in a mouse model of SARS-CoV induced disease (Agostini et al, 2018). In both prophylactic and therapeutic (24 h post infection) dosing regimens of SARS-CoV infected animals, remdesivir treated animals had a > 2 log reduction in viral titers by Day 4 or 5 post infection (Sheahan et al, 2017).…”

Third Tofo Advanced Study Week on Emerging and Re-emerging Viruses, 2018

“…The compound is now being developed by Gilead Sciences . GS-5734, a phosphoramidate prodrug of a pyrrolo [2,1-f][triazin-4-amino] adenine C-nucleoside, displays EC 50 values between 60 and 200 nM against various filoviruses in different cells including human macrophages, but is also active against arenaviruses and coronaviruses Agostini et al, 2018), while the CC 50 is > 10 μM. During the final stages of the massive 2014-2016 EVD epidemic in West Africa, the compound was succesfully used to treat EVD in a newborn child (Dörnemann et al, 2017) and a relapse in a survivor (Jacobs et al, 2016).…”