Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N4-hydroxycytidine-5′-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

Highlights d Remdesivir binding of active site of polymerase is conserved across all human CoVs d Remdesivir inhibits SARS-CoV-2 in primary and continuous human lung cell cultures d Remdesivir potency depends on cell-type-specific metabolism to its active form d Therapeutic remdesivir reduces viral loads and improves outcomes in mice

The nucleoside analog remdesivir (RDV) is a Food and Drug Administration–approved antiviral for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Thus, it is critical to understand factors that promote or prevent RDV resistance. We passaged SARS-CoV-2 in the presence of increasing concentrations of GS-441524, the parent nucleoside of RDV. After 13 passages, we isolated three viral lineages with phenotypic resistance as defined by increases in half-maximal effective concentration from 2.7- to 10.4-fold. Sequence analysis identified nonsynonymous mutations in nonstructural protein 12 RNA-dependent RNA polymerase ( nsp12 -RdRp): V166A, N198S, S759A, V792I, and C799F/R. Two lineages encoded the S759A substitution at the RdRp Ser 759 -Asp-Asp active motif. In one lineage, the V792I substitution emerged first and then combined with S759A. Introduction of S759A and V792I substitutions at homologous nsp12 positions in murine hepatitis virus demonstrated transferability across betacoronaviruses; introduction of these substitutions resulted in up to 38-fold RDV resistance and a replication defect. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a roughly 10-fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, whereas nsp12 -V792I diminished the uridine triphosphate concentration needed to overcome template-dependent inhibition associated with RDV. The in vitro–selected substitutions identified in this study were rare or not detected in the greater than 6 million publicly available nsp12 -RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in clinical settings.

1Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent 2 of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the 3 urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging 4CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, 5 potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial 6 cultures (EC50 = 0.01 µM). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 µM) due to their 7 low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-8CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice 9 infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved 10 pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is 11 potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for 12 treatment of COVID-19. 13 14 17 virus-specific (Sheahan et al., 2017). Together, these data demonstrate that RDV is potently antiviral 131 against SARS-CoV-2 in primary human lung cultures with a selectivity index of >1000. 132 Antiviral activities of RDV and GS-441524 correlate with RDV-TP metabolite levels. Cell type 133 specific expression of genes that metabolize ribonucleoside analogs can have a profound impact on 134 activity (Eriksson, 2013;Koczor et al., 2012). Table 1 and prior studies (Bojkova et al., 2020; Choy et al., 135 2020;Jeon et al., 2020) demonstrate the antiviral activity of RDV against SARS-CoV-2 is highly variable 136 in different cell culture models. Both RDV and GS-441524 undergo intracellular conversion to the active 137 metabolite RDV-TP involving several metabolic steps (Fig. S4) and the efficiency of each step might 138 differ between cell types. Therefore, to reconcile the differences in antiviral activity of RDV and GS-139 441524 observed in our and other studies, we compared intracellular RDV-TP concentrations in Vero E6, 140Calu3 2B4, and HAEs following incubation with the two compounds. RDV-TP levels per million cells