Pharmacological inhibition of bromodomain and extra-terminal proteins induces an NRF-2-mediated antiviral state that is subverted by SARS-CoV-2 infection
Fig 8
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.