Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase

Ariane J Brown, John J Won, Rachel L Graham, Kenneth H Dinnon 3rd, Amy C Sims, Joy Y Feng, Tomas Cihlar, Mark R Denison, Ralph S Baric, Timothy P Sheahan, Ariane J Brown, John J Won, Rachel L Graham, Kenneth H Dinnon 3rd, Amy C Sims, Joy Y Feng, Tomas Cihlar, Mark R Denison, Ralph S Baric, Timothy P Sheahan

Abstract

The genetically diverse Orthocoronavirinae (CoV) family is prone to cross species transmission and disease emergence in both humans and livestock. Viruses similar to known epidemic strains circulating in wild and domestic animals further increase the probability of emergence in the future. Currently, there are no approved therapeutics for any human CoV presenting a clear unmet medical need. Remdesivir (RDV, GS-5734) is a monophosphoramidate prodrug of an adenosine analog with potent activity against an array of RNA virus families including Filoviridae, Paramyxoviridae, Pneumoviridae, and Orthocoronavirinae, through the targeting of the viral RNA dependent RNA polymerase (RdRp). We developed multiple assays to further define the breadth of RDV antiviral activity against the CoV family. Here, we show potent antiviral activity of RDV against endemic human CoVs OC43 (HCoV-OC43) and 229E (HCoV-229E) with submicromolar EC50 values. Of known CoVs, the members of the deltacoronavirus genus have the most divergent RdRp as compared to SARS- and MERS-CoV and both avian and porcine members harbor a native residue in the RdRp that confers resistance in beta-CoVs. Nevertheless, RDV is highly efficacious against porcine deltacoronavirus (PDCoV). These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV.

Keywords: Broad-spectrum antivirals; Coronavirus; Emerging viruses; GS-5743; Remdesivir.

Copyright © 2019 Elsevier B.V. All rights reserved.

Figures

Fig. 1
Fig. 1
HCoV-OC43 Focus forming antiviral assay. A) HCoV-OC43 antiviral assay plate layout in Huh7 cells treated with DMSO or a dose response of RDV. B) A dose dependent reduction in viral foci is visualized through antibody staining. C) The numbers of foci are quantitated on an ImmunoSpot Elispot reader to generate % inhibition values. D) Example foci from a complete dose response of RDV. E) The quantitated number of spots per well for three independent experiments (A, B, C). Each dot represents the data from one well in a multiwell plate. The line is drawn at the mean and error bars represent the standard deviation. F) EC50 values were generated through graphing the percent inhibition from the above data in Graphpad Prism 8.
Fig. 2
Fig. 2
RNA FISH and qRT-PCR demonstrate RDV diminishes HCoV-OC43 viral RNA. (A) Photomicrographs of HCoV-OC43 genomic RNA fluorescence in situ hybridization (FISH). Huh7 infected with HCoV-OC43 at an MOI of 0.025 treated with dilutions of RDV or DMSO. Cell nuclei were counterstained with Hoechst. The bar is 100 μM. Representative pictures from two independent experiments are shown. (B) Quantitation of RNA FISH signal through the creation of signal area masks for nuclei and HCoV-OC43 FISH signals. (C) Enumeration of nuclei and HCoV-OC43 RNA FISH area in three random fields per condition. (D) Confirmation of HCoV-OC43 FISH data with qRT-PCR for viral genomic (ORF1b) and subgenomic (nucleocapsid). Parallel plates were infected and treated similarly to those in A.
Fig. 3
Fig. 3
HCoV 229E antiviral assay. A) The antiviral activity of RDV against HCoV-229E was measured in a cytopathic effect-based assay in Huh7 cells by CellTiter-Glo assay. Each dot represents the data from one well in a 96-well plate. The line is at the mean and error bars represent the standard deviation. B) Cytotoxicity of RDV was measured via CellTiter-Glo assay. C) Percent inhibition and percent cytotoxicity from data in panel A and panel B to determine EC50 and CC50. Five independent studies were performed (average EC50 = 0.024 μM). Representative data from a single experiment is shown.
Fig. 4
Fig. 4
Variation in CoV RdRp and susceptibility to RDV. (A) Dendrogram showing genetic relatedness of representative human and zoonotic CoV RdRp proteins. Font color of virus name corresponds to natural host for virus shown above. Amino acid sequence alignment shows 483L RDV resistance mutation is naturally occurring in deltacoronavirus (genotype 4). The percent similarity of each RdRp as compared to SARS-CoV and MERS-CoV is indicated in the heat map. (B) Variation in CoV specific, fingers, palm and thumb domains of RdRp shown in % amino acid identity plot (top) and in the more detailed heat map showing % identity per residue within the RdRp functional domains (A–G) and RDV resistance mutations identified in MHV (F476L, V553L) mapped. (C) Amino acid multiple sequence alignments for each RdRp functional motif showing high conservation. Amino acid positions are noted in the multiple sequence alignment which corresponds to positions above as well as in SARS-CoV RdRp (bold).
Fig. 5
Fig. 5
Porcine deltacoronavirus is susceptible to the antiviral activity of RDV. A) Overview of the CPE-based antiviral assay for PDCoV in LLC-PK1 cells. (B) Representative relative light units (RLU) raw data from one of four independent PDCoV assays in LLC-PK1 cells. C) Example cytoxicity data (RLU) measured via CellTiter-Glo for RDV in LLC-PK1 cells. (D) RDV EC50 and CC50 curves for PDCoV in LLC-PK1 cells. (E) Overview of RDV CPE-based antiviral assays in either LLC-PK1 or Huh7 cells for HCoV-229E using PDCoV media conditions. (F) RLU raw data for HCoV-229E in LLC-PK1 or Huh7 cells using media formulations for PDCoV. (G) EC50 curves for raw data shown in F. (H) Overview of RDV CPE-based antiviral assay for PDCoV in Huh7 cells. (I) Representative RLU raw data one of five independent PDCoV assays in Huh7 cells. (J) Example cytoxicity data (RLU) measured via CellTiter-Glo for RDV in Huh7 cells for assay described in “H”. (K) Representative RDV EC50 and CC50 curves for PDCoV in Huh7 cells. For B, C, F, I and J, each dot represents the data from one well in a 96-well plate. The line is at the mean and error bars represent the standard deviation.

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