Prophylactic administration of JQ-1 suppresses the expression of <i>IFIT1</i> and <i>MX2</i> mRNA levels.

(A-B) Quantification of the dose-dependent effect of DMSO and JQ-1 on the mRNA expression levels of (A) IFIT1 and (B) MX2 in Calu-3 cells. Calu-3 cells were pretreated for 48 hours with corresponding concentrations of DMSO or JQ-1 (0.04–5.12 μM) prior to infection with SARS-CoV-2 (MOI = 0.1) for 24 hours under continuous treatment. At post infection, infected cells were RNA-extracted and analysed by qRT-PCR for mRNA expression from the indicated genes. Unpaired parametric t-test with the Holm-Šídák correction for multiple testing was used to compare the means from three independent experiments. (PDF)</p

SARS-CoV-2 infection and JQ-1 treatment modulate the chromatin regulatory landscape.

(A) TSS plot depicting the density and distribution of accessible ATAC-seq peaks around transcription start sites within a window of -500 to 500 bp. (B) Analysis of the genomic features annotated to accessible ATAC-seq peaks in the chromatin. (C) Heatmap of differentially accessible ATAC-seq peaks scaled as z-score across rows. (D-G) Volcano plots showing relative log2 fold change (log2FC) and statistical significance [-log10 (p-value)] of differentially accessible ATAC-seq peaks in (D) DMSO-treated infected/DMSO-treated uninfected, (E) JQ-1-treated uninfected/DMSO-treated uninfected, (F) JQ-1-treated infected/DMSO-treated infected, and (G) JQ-1-treated infected/JQ-1-treated uninfected contrasts. Significantly (FDR of ≤0.05) regulated peaks are indicated. (H-K) TF motif enrichment of accessible ATAC-seq peaks in (H) DMSO-treated infected/DMSO-treated uninfected, (I) JQ-1-treated uninfected/DMSO-treated uninfected, (J) JQ-1-treated infected/DMSO-treated infected and (K) JQ-1-treated infected/JQ-1-treated uninfected contrasts. The TF motifs were identified using the DREME algorithm, where the height of the letters represents the frequency of each base in the motif.</p

Prophylactic administration of iBETs inhibits SARS-CoV-2 infection.

(A-B) Dose response curves (n = 3) showing the effect of the indicated iBETs on the quantities of (A) SARS-CoV-2 genomic RNA (E copies/μl) and (B) infectious titers (PFU/ml) in the supernatant at 24 h.p.i. Calu-3 cells were pretreated for 48 hours prior to infection with SARS-CoV-2 (MOI = 0.1) for 24 hours under continuous presence of the drug. (C-D) Relative quantification of SARS-CoV-2 (C) genomic RNA (E copies/μl) and (D) titers (PFU/ml) in the supernatants harvested from the apical compartment of hBAECs at 48 h.p.i. Cultures were pretreated with DMSO or JQ-1 (2.56 μM) for 48 hours and infected with SARS-CoV-2 (2x104 PFUs) for 48 hours under continuous presence of the drug. Unpaired parametric t-test was used to compare the means from duplicates of five and four independent experiments, respectively. (E) Representative FACS dot plots and (F) relative quantification of GFP-positive cells in cell populations that were treated with DMSO or JQ-1 (2.56 μM) prior to infection with SARS-CoV-2-GFP (MOI = 0.25). Calu-3 cells were pretreated for 48 hours prior to infection with SARS-CoV-2 (MOI = 0.1) for 24 hours under continuous presence of the drug. GFP expression was quantified by flow cytometry and the data are shown as GFP-positive cells from treated cells relative to untreated cells. Unpaired parametric t-test was used to compare the means from triplicates of three independent experiments. Raw data are shown in S1 Data.</p

SARS-CoV-2 infection and JQ-1 treatment modulate the transcriptomic and proteomic profiles.

(A) PCA of significantly expressed genes showing the variance of gene expression profiles between experimental groups. Each symbol represents a technical replicate (B) Dot plot of GO biological pathway terms showing pathway enrichment from differentially expressed genes (DRGs) between indicated contrasts. Pathways were selected by filtering for the top 15 pathways with the largest (absolute value) normalised enrichment score (NES) per contrast. (C) Bio-Safe Coomassie Blue staining of proteins from Calu-3 cell lysates resolved on linear (7.5%) SDS-PAGE gels prior to mass spectrometry analysis. (D) PCA of significantly abundant proteins showing the variance of protein profiles between experimental groups. Each symbol represents a technical replica. (PDF)</p

Raw data corresponding to the figure panels showing normalizations.

Raw data corresponding to the figure panels showing normalizations.</p

SARS-CoV-2 and JQ-1-mediated modulations of the chromatin accessibility landscape modulate biological pathways.

(A) PCA of significantly accessible ATAC-seq peaks showing the variance of peak accessibility profiles between experimental groups. Each symbol represents a technical replicate (B-C) Dot plots of GO biological pathway terms from genes annotated to the ATAC-seq peaks with significantly (B) increased and (C) decreased accessibilities between indicated contrasts. Dot colour represents normalised enrichment score (NES) and dot size (peak ratio) represents the number of significant peaks related to the GO biological pathway term relative to the total number of significant peaks. (PDF)</p

SARS-CoV-2 and JQ-1-mediated modulations of the chromatin accessibility landscape underlie changes in transcriptomic and proteomic profiles.

(A-D) Volcano plots showing relative log2FC and statistical significance [-log10 (p-value)] of differentially regulated genes (DRGs) in (A) DMSO-treated infected/DMSO-treated uninfected, (B) JQ-1-treated uninfected/DMSO-treated uninfected, (C) JQ-1-treated infected/DMSO-treated infected, (D) JQ-1-treated infected/JQ-1-treated uninfected contrasts. The numbers of significant (FDR of ≤0.05) DRGs are indicated. (E) Heatmap of DRGs across four biological pathways (IFN-stimulated genes, cell cycle-regulating genes, autophagy-regulating genes, and NRF-2-regulated genes) scaled as z-score across rows. (F-I) Volcano plots showing relative log2FC and statistical significance [-log10 (p-value)] of differentially abundant proteins in (F) DMSO-treated infected/DMSO-treated uninfected, (G) JQ-1-treated uninfected/DMSO-treated uninfected, (H) JQ-1-treated infected/DMSO-treated infected and (I) JQ-1-treated infected/JQ-1-treated uninfected contrasts. The numbers of significantly (FDR ≤0.05) regulated proteins are indicated. (J-N) Top enriched (FDR ≤0.05) GO biological pathway terms of upregulated proteins in (J) DMSO-treated infected/DMSO-treated uninfected, and differentially regulated proteins in (K-L) JQ-1-treated uninfected/DMSO-treated uninfected and (M-N) JQ-1-treated infected/DMSO-treated infected contrasts.</p

S2 Table -

Codons mapped to SARS-CoV-2 ORF6 between positions 27217–27219 in the genome of (A) input SARS-CoV-2 and (B) serially passaged (P15) SARS-CoV-2 virions under increasing concentrations of DMSO and JQ-1 in Calu-3 cells. The stop codon is marked with an asterisk. (PDF)</p

SARS-CoV-2 infection modulates abundance of proteins involved in oxidative phosphorylation, chromatin condensation, and epitranscriptome.

(A) Log2FC analysis of differentially abundant proteins implicated in oxidative phosphorylation (COQ6 and COX16), chromatin silencing (H2AFY, H2AFV & H2AFZ), and epitranscriptomics (YTHDF2) in indicated contrasts. The bars indicate relative log2FC in protein abundance between experimental groups in each contrast, and proteins with a relative log2FC of 1 and an FDR≤0.05 were considered significant. (PDF)</p

SARS-CoV-2 subverts JQ-1-mediated antiviral activity.

(A) Quantification of SARS-CoV-2 RNA (E copies/μl) from 15 serial passages under two-fold escalating concentrations of JQ-1, with DMSO as a mock. Unpaired parametric t-test with the Holm-Šídák correction for multiple testing was used to compare the means from triplicates of one experiment. (B) Quantification of SARS-CoV-2 titers (PFU/ml) in triplicates from P1 (JQ-1 0.32μM) and P15 (JQ-1 5.12μM) passages under JQ-1 selection. Unpaired parametric t-test with the Holm-Šídák correction for multiple testing was used to compare the means from triplicates of one experiment. Quantification of (C) IC50 and (D) IC90 values from JQ-1 dose-dependent inhibition curves determined in JQ-1-treated Calu-3 cells infected with parental and passaged SARS-CoV-2 virions (MOI = 0.1) based on viral RNA concentrations in the supernatant (S6C Fig). One-way ANOVA with Dunnett’s multiple comparison test was used to compare the means from three independent experiments with the parental virus and four independent experiments with passaged SARS-CoV-2 virions. (E) Representative plaque phenotypes derived in Vero E6 cells showing plaque morphologies from passaged SARS-CoV-2 virions following infection of IFN-treated Calu-3 cells. (F) Viral growth kinetics in DMSO-treated Calu-3 cells showing viral RNA quantities (E copies/μl) from parental and passaged (P15) SARS-CoV-2 virions (MOI = 0.1). Unpaired parametric t-test with the Holm-Šídák correction for multiple testing was used to compare the means from four independent experiments. (G) Schematic diagram of the SARS-CoV-2 genome depicting the premature stop codon at position six of ORF6 in sequences derived from the passaged (P15) virions. (H) Quantification of ORF6 D6* mutation frequency from sequences generated from the genome of passaged (P15) SARS-CoV-2 virions. Immunoblot analysis of SARS-CoV-2-ORF6 and nucleocapsid (N) expression in Calu-3 cells infected with DMSO-selected and JQ-1-selected SARS-CoV-2 (P15, MOI = 0.1). (I) Quantification of SARS-CoV-2 genomic RNA (E copies/μl) from passaged (P15) SARS-CoV-2 virions in the supernatants of IFN-treated Calu-3 cells at 24 h.p.i. Calu-3 cells were pretreated for 24 hours with indicated concentrations of IFNα-2a prior to infection with SARS-CoV-2 under continuous IFN treatment. Unpaired parametric t-test with the Holm-Šídák correction for multiple testing was used to compare the means from duplicates of two independent experiments. (J-K) Virus growth kinetics in DMSO and JQ-1 (2.56 μM)-treated hBAECs depicting SARS-CoV-2 RNA quantities (E copies/μl) in the culture supernatants following (J) prophylactic and (K) therapeutic drug administration. (L-M) Virus growth kinetics in DMSO and JQ-1 (2.56 μM)-treated hBAECs depicting SARS-CoV-2 infectious titers (PFU/ml) in the culture supernatants following following (L) prophylactic and (M) therapeutic drug administration. The area shaded in red indicates the time period of drug administration and the grey lines in the context of therapeutic drug administration indicate infection before drug administration. Unpaired parametric t-test with the Holm-Šídák correction for multiple testing was used to compare the means from triplicates of one experiment. Raw data are shown in S1 Data.</p