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RESEARCH ARTICLE
CORONAVIRUS
An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice
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Science Translational Medicine 29 Apr 2020:
Vol. 12, Issue 541, eabb5883
DOI: 10.1126/scitranslmed.abb5883Catastrophic consequences
Broad-spectrum antivirals are desirable, particularly in the context of emerging zoonotic infections for which specific interventions do not yet exist. Sheahan et al. tested the potential of a ribonucleoside analog previously shown to be active against other RNA viruses such as influenza and Ebola virus to combat coronaviruses. This drug was effective in cell lines and primary human airway epithelial cultures against multiple coronaviruses including SARS-CoV-2. Mouse models of SARS and MERS demonstrated that early treatment reduced viral replication and damage to the lungs. Mechanistically, this drug is incorporated into the viral RNA, inducing mutations and eventually leading to error catastrophe in the virus. In this manner, inducing catastrophe could help avoid catastrophe by stemming the next pandemic.
Abstract
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.
INTRODUCTION
The genetically diverse Orthocoronavirinae [coronavirus (CoV)] family circulates in many avian and mammalian species. Phylogenetically, CoVs are divided into four genera: alpha (group 1), beta (group 2), gamma (group 3), and delta (group 4). Three new human CoV have emerged in the past 20 years with severe acute respiratory syndrome CoV (SARS-CoV) in 2002, Middle East respiratory syndrome CoV (MERS-CoV) in 2012, and now SARS-CoV-2 in 2019 (1–3). All human CoV are thought to have emerged originally as zoonoses (4–6).
The ongoing SARS-CoV-2 pandemic [referred to as CoV disease 2019 (COVID-19)] has caused more than 500,000 infections and more than 25,000 deaths in 199 countries. Like SARS-CoV and MERS-CoV, the respiratory disease caused by SARS-CoV-2 can progress to acute lung injury (ALI), an end-stage lung disease with limited treatment options and very poor prognoses (3, 7, 8). This emergence paradigm is not limited to humans. A novel group 1 CoV called swine acute diarrhea syndrome CoV (SADS-CoV) recently emerged from bats causing the loss of more than 20,000 pigs in Guangdong Province, China (9). More alarmingly, many group 2 SARS-like and MERS-like CoVs are circulating in bat reservoir species that can use human receptors and replicate efficiently in primary human lung cells without adaptation (9–12).
The presence of these “preepidemic” zoonotic strains foreshadow the emergence and epidemic potential of additional SARS-like and MERS-like viruses in the future. Given the diversity of CoV strains in zoonotic reservoirs and a penchant for emergence, broadly active antivirals are clearly needed for rapid response to new CoV outbreaks in humans and domesticated animals.
Currently, there are no approved therapies specific for any human CoV. β-D-N4-hydroxycytidine (NHC; EIDD-1931) is an orally bioavailable ribonucleoside analog with broad-spectrum antiviral activity against various unrelated RNA viruses including influenza, Ebola, CoV, and Venezuelan equine encephalitis virus (VEEV) (13–16). For VEEV, the mechanism of action (MOA) for NHC has been shown to be through lethal mutagenesis where deleterious transition mutations accumulate in viral RNA (14, 17). Thus, we sought to determine NHC’s breadth of antiviral activity against multiple emerging CoV, its MOA for CoV, and its efficacy in mouse models of CoV pathogenesis.
RESULTS
NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication
To determine whether NHC blocks the replication of highly pathogenic human CoV, we performed antiviral assays in cell lines with MERS-CoV and the newly emerging SARS-CoV-2. We first assessed the antiviral activity of NHC against MERS-CoV in the human lung epithelial cell line Calu-3 2B4 (“Calu-3” cells). Using a recombinant MERS-CoV expressing nanoluciferase (MERS-nLUC) (18), we measured virus replication in cultures exposed to a dose range of drug for 48 hours. NHC was potently antiviral with an average median inhibitory concentration (IC50) of 0.15 μM and no observed cytoxicity in similarly treated uninfected cultures across the dose range [50% cytotoxic concentration (CC50) of >10 μM; Fig. 1A]. The therapeutic index for NHC was >100. Using a clinical isolate of SARS-CoV-2 (2019-nCoV/USA-WA1/2020), we performed antiviral assays in African green monkey kidney (Vero) cells and found that NHC was potently antiviral with an IC50 of 0.3 μM and CC50 of >10 μM (Fig. 1B). We then determined the antiviral activity of NHC against SARS-CoV-2 in the Calu-3 cells through the measurement of infectious virus production and viral genomes. We observed a dose-dependent reduction in virus titers (Fig. 1C) with an IC50 of 0.08 μM. Viral genomic RNA was quantitated in clarified supernatants by quantitative reverse transcription polymerase chain reaction (qRT-PCR; Fig. 1D). Like the effect on infectious titers, we found a dose-dependent reduction in viral genomic RNA and a similar calculated IC50 of 0.09 μM. Collectively, these data demonstrate that NHC is potently antiviral against two genetically distinct emerging CoV.
Fig. 1 NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication.
(A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (MERS-nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via CellTiter-Glo assay. Data are combined from three independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by CellTiter-Glo assay. Cytotoxicity was measured as in (A). Data are combined from two independent experiments. (C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with SARS-CoV-2 at an MOI of 0.1 for 30 min, washed, and exposed to a dose response of NHC in triplicate per condition. At 72 hpi, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in (C). Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For (A) to (D), the symbol is at the mean, and the error bars represent the SD.
NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures
To determine whether NHC would be similarly antiviral in primary human cells, we performed a series of studies in primary human airway epithelia (HAE) cell cultures. HAE models the architecture and cellular complexity of the conducting airway and is readily infected by multiple human and zoonotic CoV, including SARS-CoV and MERS-CoV (19). We first assessed the cytotoxicity of NHC in HAE treated with an extended dose range for 48 hours using quantitative PCR of cell death–related gene transcripts as our metric. NHC treatment did not appreciably alter gene expression even at doses up to 100 μM (fig. S1). We then sought to determine whether NHC would inhibit clinical isolate SARS-CoV-2 replication in HAE. We observed a dose-dependent reduction in SARS-CoV-2 infectious virus production (Fig. 2A). In MERS-CoV–infected HAE, NHC substantially reduced virus production with maximal titer reduction of >5 logs at 10 μM (average IC50 = 0.024 μM), which correlated with reduced genomic open reading frame 1 (ORF1) and subgenomic [ORF nucleocapsid (ORFN)] RNA in paired samples (Fig. 2B). We observed similar trends in titer reduction (>3 log at 10 μM, average IC50 = 0.14 μM) and in copies of genomic and subgenomic RNA in SARS-CoV–infected HAE (Fig. 2C). Thus, NHC was potently antiviral against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human epithelial cell cultures without cytotoxicity.
Fig. 2 NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary HAE cell cultures.
(A) Human airway epithelia (HAE) cultures were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate, after which the virus was removed, and cultures were washed in, incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to (A). qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in (C) for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units; l.o.d., limit of detection. (C) Studies performed as in (A) but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean, and the error bars represent the SD.
NHC is effective against remdesivir-resistant virus and multiple distinct zoonotic CoV
CoV are taxonomically divided into multiple genogroups (alpha, beta, gamma, and delta), but human-infecting CoV are found only in the alpha and beta subgroups thus far (Fig. 3A). There is high sequence conservation in the RNA-dependent RNA polymerase [RdRp; nonstructural protein 12 (nsp12)] across CoV (Fig. 3A). For example, the RdRp of SARS-CoV-2 has 99.1% similarity and 96% amino acid identity to that of SARS-CoV (Fig. 3A). To gain insight into structural conservation of RdRp across the CoV family, we modeled the variation reflected in the RdRp dendrogram in Fig. 3A onto the structure of the SARS-CoV RdRp (Fig. 3B) (20). The core of the RdRp molecule and main structural motifs that all RdRp harbor (Fig. 3B and fig. S2) is highly conserved among CoV including SARS-CoV-2. We previously reported that CoV resistance to another broad-spectrum nucleoside analog, remdesivir (RDV), was mediated by RdRp residues F480L and V557L in a model CoV mouse hepatitis virus (MHV) and in SARS-CoV, resulting in a fivefold shift in IC50 (Fig. 3C) (21). Consequently, we tested whether RDV resistance mutations in MHV conferred cross-re.....
https://stm.sciencemag.org/content/12/541/eabb5883
CORONAVIRUS
An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice
- Timothy P. Sheahan1,*,†,
- Amy C. Sims1,*,‡,
- Shuntai Zhou2,
- Rachel L. Graham1,
- Andrea J. Pruijssers3,
- Maria L. Agostini3,
- Sarah R. Leist1,
- Alexandra Sch?fer1,
- Kenneth H. Dinnon III1,4,
- Laura J. Stevens3,
- James D. Chappell3,
- Xiaotao Lu3,
- Tia M. Hughes3,
- Amelia S. George3,
- Collin S. Hill2,
- Stephanie A. Montgomery5,
- Ariane J. Brown1,
- Gregory R. Bluemling6,7,
- Michael G. Natchus6,
- Manohar Saindane6,
- Alexander A. Kolykhalov6,7,
- George Painter6,7,8,
- Jennifer Harcourt9,
- Azaibi Tamin9,
- Natalie J. Thornburg9,
- Ronald Swanstrom2,10,
- Mark R. Denison3 and
- Ralph S. Baric1,4,†
See all authors and affiliations
Science Translational Medicine 29 Apr 2020:
Vol. 12, Issue 541, eabb5883
DOI: 10.1126/scitranslmed.abb5883Catastrophic consequences
Broad-spectrum antivirals are desirable, particularly in the context of emerging zoonotic infections for which specific interventions do not yet exist. Sheahan et al. tested the potential of a ribonucleoside analog previously shown to be active against other RNA viruses such as influenza and Ebola virus to combat coronaviruses. This drug was effective in cell lines and primary human airway epithelial cultures against multiple coronaviruses including SARS-CoV-2. Mouse models of SARS and MERS demonstrated that early treatment reduced viral replication and damage to the lungs. Mechanistically, this drug is incorporated into the viral RNA, inducing mutations and eventually leading to error catastrophe in the virus. In this manner, inducing catastrophe could help avoid catastrophe by stemming the next pandemic.
Abstract
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.
INTRODUCTION
The genetically diverse Orthocoronavirinae [coronavirus (CoV)] family circulates in many avian and mammalian species. Phylogenetically, CoVs are divided into four genera: alpha (group 1), beta (group 2), gamma (group 3), and delta (group 4). Three new human CoV have emerged in the past 20 years with severe acute respiratory syndrome CoV (SARS-CoV) in 2002, Middle East respiratory syndrome CoV (MERS-CoV) in 2012, and now SARS-CoV-2 in 2019 (1–3). All human CoV are thought to have emerged originally as zoonoses (4–6).
The ongoing SARS-CoV-2 pandemic [referred to as CoV disease 2019 (COVID-19)] has caused more than 500,000 infections and more than 25,000 deaths in 199 countries. Like SARS-CoV and MERS-CoV, the respiratory disease caused by SARS-CoV-2 can progress to acute lung injury (ALI), an end-stage lung disease with limited treatment options and very poor prognoses (3, 7, 8). This emergence paradigm is not limited to humans. A novel group 1 CoV called swine acute diarrhea syndrome CoV (SADS-CoV) recently emerged from bats causing the loss of more than 20,000 pigs in Guangdong Province, China (9). More alarmingly, many group 2 SARS-like and MERS-like CoVs are circulating in bat reservoir species that can use human receptors and replicate efficiently in primary human lung cells without adaptation (9–12).
The presence of these “preepidemic” zoonotic strains foreshadow the emergence and epidemic potential of additional SARS-like and MERS-like viruses in the future. Given the diversity of CoV strains in zoonotic reservoirs and a penchant for emergence, broadly active antivirals are clearly needed for rapid response to new CoV outbreaks in humans and domesticated animals.
Currently, there are no approved therapies specific for any human CoV. β-D-N4-hydroxycytidine (NHC; EIDD-1931) is an orally bioavailable ribonucleoside analog with broad-spectrum antiviral activity against various unrelated RNA viruses including influenza, Ebola, CoV, and Venezuelan equine encephalitis virus (VEEV) (13–16). For VEEV, the mechanism of action (MOA) for NHC has been shown to be through lethal mutagenesis where deleterious transition mutations accumulate in viral RNA (14, 17). Thus, we sought to determine NHC’s breadth of antiviral activity against multiple emerging CoV, its MOA for CoV, and its efficacy in mouse models of CoV pathogenesis.
RESULTS
NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication
To determine whether NHC blocks the replication of highly pathogenic human CoV, we performed antiviral assays in cell lines with MERS-CoV and the newly emerging SARS-CoV-2. We first assessed the antiviral activity of NHC against MERS-CoV in the human lung epithelial cell line Calu-3 2B4 (“Calu-3” cells). Using a recombinant MERS-CoV expressing nanoluciferase (MERS-nLUC) (18), we measured virus replication in cultures exposed to a dose range of drug for 48 hours. NHC was potently antiviral with an average median inhibitory concentration (IC50) of 0.15 μM and no observed cytoxicity in similarly treated uninfected cultures across the dose range [50% cytotoxic concentration (CC50) of >10 μM; Fig. 1A]. The therapeutic index for NHC was >100. Using a clinical isolate of SARS-CoV-2 (2019-nCoV/USA-WA1/2020), we performed antiviral assays in African green monkey kidney (Vero) cells and found that NHC was potently antiviral with an IC50 of 0.3 μM and CC50 of >10 μM (Fig. 1B). We then determined the antiviral activity of NHC against SARS-CoV-2 in the Calu-3 cells through the measurement of infectious virus production and viral genomes. We observed a dose-dependent reduction in virus titers (Fig. 1C) with an IC50 of 0.08 μM. Viral genomic RNA was quantitated in clarified supernatants by quantitative reverse transcription polymerase chain reaction (qRT-PCR; Fig. 1D). Like the effect on infectious titers, we found a dose-dependent reduction in viral genomic RNA and a similar calculated IC50 of 0.09 μM. Collectively, these data demonstrate that NHC is potently antiviral against two genetically distinct emerging CoV.
Fig. 1 NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication.(A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (MERS-nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via CellTiter-Glo assay. Data are combined from three independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by CellTiter-Glo assay. Cytotoxicity was measured as in (A). Data are combined from two independent experiments. (C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with SARS-CoV-2 at an MOI of 0.1 for 30 min, washed, and exposed to a dose response of NHC in triplicate per condition. At 72 hpi, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in (C). Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For (A) to (D), the symbol is at the mean, and the error bars represent the SD.
NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures
To determine whether NHC would be similarly antiviral in primary human cells, we performed a series of studies in primary human airway epithelia (HAE) cell cultures. HAE models the architecture and cellular complexity of the conducting airway and is readily infected by multiple human and zoonotic CoV, including SARS-CoV and MERS-CoV (19). We first assessed the cytotoxicity of NHC in HAE treated with an extended dose range for 48 hours using quantitative PCR of cell death–related gene transcripts as our metric. NHC treatment did not appreciably alter gene expression even at doses up to 100 μM (fig. S1). We then sought to determine whether NHC would inhibit clinical isolate SARS-CoV-2 replication in HAE. We observed a dose-dependent reduction in SARS-CoV-2 infectious virus production (Fig. 2A). In MERS-CoV–infected HAE, NHC substantially reduced virus production with maximal titer reduction of >5 logs at 10 μM (average IC50 = 0.024 μM), which correlated with reduced genomic open reading frame 1 (ORF1) and subgenomic [ORF nucleocapsid (ORFN)] RNA in paired samples (Fig. 2B). We observed similar trends in titer reduction (>3 log at 10 μM, average IC50 = 0.14 μM) and in copies of genomic and subgenomic RNA in SARS-CoV–infected HAE (Fig. 2C). Thus, NHC was potently antiviral against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human epithelial cell cultures without cytotoxicity.
Fig. 2 NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary HAE cell cultures.(A) Human airway epithelia (HAE) cultures were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate, after which the virus was removed, and cultures were washed in, incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to (A). qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in (C) for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units; l.o.d., limit of detection. (C) Studies performed as in (A) but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean, and the error bars represent the SD.
NHC is effective against remdesivir-resistant virus and multiple distinct zoonotic CoV
CoV are taxonomically divided into multiple genogroups (alpha, beta, gamma, and delta), but human-infecting CoV are found only in the alpha and beta subgroups thus far (Fig. 3A). There is high sequence conservation in the RNA-dependent RNA polymerase [RdRp; nonstructural protein 12 (nsp12)] across CoV (Fig. 3A). For example, the RdRp of SARS-CoV-2 has 99.1% similarity and 96% amino acid identity to that of SARS-CoV (Fig. 3A). To gain insight into structural conservation of RdRp across the CoV family, we modeled the variation reflected in the RdRp dendrogram in Fig. 3A onto the structure of the SARS-CoV RdRp (Fig. 3B) (20). The core of the RdRp molecule and main structural motifs that all RdRp harbor (Fig. 3B and fig. S2) is highly conserved among CoV including SARS-CoV-2. We previously reported that CoV resistance to another broad-spectrum nucleoside analog, remdesivir (RDV), was mediated by RdRp residues F480L and V557L in a model CoV mouse hepatitis virus (MHV) and in SARS-CoV, resulting in a fivefold shift in IC50 (Fig. 3C) (21). Consequently, we tested whether RDV resistance mutations in MHV conferred cross-re.....
https://stm.sciencemag.org/content/12/541/eabb5883