Fig. 3
From: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
REV-ERBα inhibits hepatitis C virus (HCV) RNA replication. a Silencing Rev-erbα increases HCV replication. Huh-7 cells supporting a HCV JFH-1-LUC replicon were transduced with lentivirus encoding shRev-erbα or control and silencing confirmed by measuring Rev-erbα mRNA and protein expression levels (mean ± S.E.M., n = 4, Mann–Whitney test). Densitometric analysis quantified REV-ERB in individual samples and was normalised to its own GAPDH loading control. HCV replication-dependent reporter activity was measured and expressed relative to control (shCtrl) cells (mean ± S.E.M., n = 6, Mann–Whitney test). b Anti-viral activity of SR9009 agonist is dependent on REV-ERB expression levels. shRev-eRbα and Ctrl HCV JFH-1 replicon cells described in (a) were treated with REV-ERB agonist SR9009 for 24 h, viral replication measured and the concentration of agonist required to inhibit viral replication by 50% defined (IC50) (mean ± S.E.M., n = 3). c REV-ERBα overexpression inhibits HCV RNA replication. Huh-7 cells stably supporting a HCV JFH-1-LUC replicon were transfected with empty plasmid or vector expressing REV-ERBα and 48 h later protein expression assessed by western blotting and viral replication measured (mean ± S.E.M., n = 4, Mann–Whitney statistical test). Data are plotted relative to Ctrl untreated cells. d REV-ERB agonists cure HCV-infected cells. HCVcc SA13/JFH-1 infected Huh-7 cells were treated with increasing concentrations of REV-ERB agonists for 24 h and viral RNA or NS5A-expressing cells quantified and data expressed relative to Ctrl untreated cells. The experiment was performed in the presence of a neutralising anti-CD81 antibody to limit secondary rounds of infection (mean ± S.E.M., n = 3). e REV-ERB ligands inhibit the replication of diverse HCV genotypes. Huh-7 cells transiently supporting HCV sub-genomic replicons representing genotypes 1–3 were treated with the REV-ERB agonists SR9009 or GSK2667 and replication assessed 24 h later. The dose of agonist required to inhibit HCV RNA replication by 50% (IC50) was determined for all viral genotypes (mean ± S.E.M., n = 3)
