The Inhaled Steroid Ciclesonide Blocks SARS-CoV-2 RNA Replication by Targeting the Viral Replication-Transcription Complex in Cultured Cells

Abstract

Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and Middle East respiratory syndrome coronavirus (MERS-CoV) replication. Ciclesonide, an inhaled corticosteroid, suppressed the replication of MERS-CoV and other coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), in cultured cells. The 90% effective concentration (EC₉₀) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 μM. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in nonstructural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed the replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under a microscope, the viral RNA replication-transcription complex in cells, which is thought to be detectable using antibodies specific for nsp3 and double-stranded RNA, was observed to fall in the presence of ciclesonide in a concentration-dependent manner. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients.IMPORTANCE The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. New and effective antiviral agents that combat the disease are needed urgently. Here, we found that an inhaled corticosteroid, ciclesonide, suppresses the replication of coronaviruses, including betacoronaviruses (murine hepatitis virus type 2 [MHV-2], MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alphacoronavirus (human coronavirus 229E [HCoV-229E]), in cultured cells. Ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS and COVID-19.

Keywords: COVID-19; DMV; RTC; SARS-CoV-2; ciclesonide; coronavirus; steroid.

FIG 1

Steroid compounds reduce death rates of MERS-CoV-infected cells. (a) Cell survival. Vero cells seeded in 96-well microplates were infected with 100 50% tissue culture infective doses (TCID₅₀) of MERS-CoV in the presence of steroid compounds (10 μM). Cytopathic effects were observed at 72 h postinfection. Surviving cells were stained with crystal violet, photographed, and quantified using ImageJ software. Data are presented as the averages from two independent wells. Arrows indicate the steroid compounds assessed further in this study. (b) Four steroid compounds. The structures of the steroid compounds that conferred a >95% cell survival rate are depicted.

FIG 2

Steroid compounds suppress the replication of MERS-CoV and other viruses. (a) Effects of eight steroid compounds on MERS-CoV replication. Vero cells were infected with MERS-CoV at an MOI of 0.01 in the presence of the indicated steroids for 24 h. The viral titer in the cell supernatant was quantified by a plaque assay using Vero/TMPRSS2 cells. Cell viability in the absence of virus was quantified by a WST assay. (b) Antiviral effects of steroid compounds on various viral species. Cells were infected with the indicated viruses at an MOI of 0.01 in the presence of dimethyl sulfoxide (DMSO) (control) or the indicated steroids. The viral yield in the cell supernatant was quantified by a plaque assay or real-time PCR. Hep-2 cells were incubated with respiratory syncytial virus A (RSV-A long) for 1 day; MDCK cells were incubated with influenza virus H3N2 for 1 day; Vero cells were incubated with rubella virus (TO336) for 7 days; DBT cells were incubated with murine coronavirus (MHV-2) for 1 day; Vero cells were incubated with MERS-CoV (EMC), SARS-CoV (Frankfurt-1), or SARS-CoV-2 (WK-521) for 1 day; and HeLa229 cells were incubated with HCoV-229E (VR-740) for 1 day. Data are presented as the means ± standard deviations from four independent wells. *, P ≤ 0.05; ***, P ≤ 0.001.

FIG 3

Time-of-addition assays for MERS-CoV replication inhibitors. The inhibitor E64d, ciclesonide, or lopinavir (each at 10 μM) or DMSO (control) was added to Vero cells at the indicated times after virus inoculation (MOI of 1). The amount of cellular viral mRNA at 6 h postinfection was measured by real-time PCR using a upE primer/probe set. Data are presented as the means ± SD from 4 independent experiments.

FIG 4

A ciclesonide escape mutant of MERS-CoV. (a) Viral growth of a ciclesonide escape mutant of MERS-CoV. Vero cells treated with 10 μM ciclesonide were infected with parental MERS-CoV or the ciclesonide escape mutant at an MOI of 0.01. The viral titer in the culture medium was quantified at 24 postinfection (hpi). (b) Viral RNA replication of a ciclesonide escape mutant of MERS-CoV. Vero cells treated with 10 μM ciclesonide were infected with parental MERS-CoV or the ciclesonide escape mutant at an MOI of 1. The viral RNA in the cells was quantified at 6 hpi. E64d (10 μM), a virus entry inhibitor, was used for comparison. (c) Growth of the recombinant virus. Vero cells were infected with the parental MERS-CoV/EMC strain (Re-EMC/MERS) or the recombinant mutant strain (Re-Nsp15-A25V) containing an amino acid substitution at A25V in nsp15 at an MOI of 0.01 and then treated with the indicated compounds (10 μM). The virus titer was quantified at 24 hpi. (d) RNA replication of the recombinant virus. Vero cells were infected with Re-EMC/MERS or Re-Nsp15-A25V at an MOI of 1 and treated with the indicated compounds (10 μM). The viral RNA in infected cells was quantified at 6 hpi. ND, not detected.

FIG 5

Ciclesonide suppresses the replication of SARS-CoV-2. (a, c, and e) Time course of SARS-CoV-2 propagation. (b, d, and f) Concentration-dependent effects of ciclesonide. VeroE6/TMPRSS2 cells (a and b), Calu-3 cells (c and d), or HBTE/ALI cells (e and f) were infected with SARS-CoV-2 at an MOI of 0.001 in the presence of DMSO or ciclesonide (10 μM) and then incubated for 1, 3, or 5 days. The virus titer in medium was quantified by a plaque assay using VeroE6/TMPRSS2 cells (n = 2 [a and c]); alternatively, the viral RNA in cells or culture medium was quantified by real-time PCR using the E gene primer/probe set (n = 1 [e] or n = 4 [f]). Average cell viability in the absence of virus was quantified using a WST assay (n = 2 [b and d]).

FIG 6

Steroid compounds and other inhibitors suppress SARS-CoV-2 RNA replication in VeroE6/TMPRSS2 cells. (a) Time course of SARS-CoV-2 RNA replication. Cells were infected with virus at an MOI of 1, and cellular RNA was collected at the indicated time points. (b) Inhibition of viral RNA replication. Cells were infected with SARS-CoV-2 at an MOI of 1 in the presence of the indicated compounds (10 μM) for 6 h. Cellular viral RNA was quantified by real-time PCR using the E gene primer/probe set. ***, P ≤ 0.001.

FIG 7

A ciclesonide escape mutant of SARS-CoV-2. VeroE6/TMPRSS2 cells treated with the indicated compounds (each at 10 μM) were infected with parental SARS-CoV-2 or with the ciclesonide escape mutant (MOI = 1). Viral RNA titers in cells were measured at 6.5 hpi. Data are presented as the means ± SD from 4 independent experiments. ***, P ≤ 0.001.

FIG 8

Amino acid substitutions in ciclesonide escape mutants of SARS-CoV-2. (a) Virus replication in the presence of ciclesonide (10 μM) is due to amino acid substitutions in nsp3 and nsp4. The replication of RNA derived from the 15 mutants listed in Table 1 was assessed in VeroE6/TMPRSS2 cells. Viral RNA was isolated at 6 hpi and measured by real-time PCR using the E gene primer/probe set. The results were compared with those for the parental virus in which the viral RNA level after treatment with DMSO was set to 1 and that after treatment with nelfinavir was set to 1/1,000. Relative reductions of viral RNA in the presence of ciclesonide were plotted at the corresponding mutations in the SARS-CoV-2 genome sequence. Data are presented as the averages from two independent experiments. The amino acid substitutions in nsp3 and nsp4 are shown at the bottom. (b) Topological diagram. The C-terminal region of nsp3 and full-length nsp4 are depicted on the lipid bilayer of the endoplasmic reticulum membrane.

FIG 9

Distribution of nsp3 and double-strand RNA in the presence or absence of ciclesonide. VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at an MOI of 0.1 in the presence of DMSO or ciclesonide and then incubated for 5 h. Next, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Tween 20. nsp3 and double-strand RNA were stained with a rabbit anti-SARS-nsp4 antibody and a mouse anti-dsRNA antibody, followed by Alexa Fluor 594-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG. Cell nuclei were stained with DAPI.