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REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. <t>HCV</t> A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV <t>RNA</t> replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)
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1) Product Images from "The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication"

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

Journal: Nature Communications

doi: 10.1038/s41467-019-08299-7

REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)
Figure Legend Snippet: REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)

Techniques Used: Activation Assay, Expressing, Microarray, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Inhibition, MANN-WHITNEY

Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles
Figure Legend Snippet: Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

Techniques Used: Expressing, Over Expression

HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)
Figure Legend Snippet: HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)
Figure Legend Snippet: 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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

Techniques Used: Transduction, Expressing, MANN-WHITNEY, Activity Assay, Concentration Assay, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Infection

2) Product Images from "The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication"

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

Journal: Nature Communications

doi: 10.1038/s41467-019-08299-7

REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d REV-ERBs bind SCD1 promoter. Analyses of available ChIP-seq data in mouse livers 30 demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)
Figure Legend Snippet: REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d REV-ERBs bind SCD1 promoter. Analyses of available ChIP-seq data in mouse livers 30 demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)

Techniques Used: Activation Assay, Expressing, Microarray, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Inhibition, MANN-WHITNEY

Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles
Figure Legend Snippet: Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

Techniques Used: Expressing, Over Expression

HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)
Figure Legend Snippet: HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)
Figure Legend Snippet: 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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

Techniques Used: Transduction, Expressing, MANN-WHITNEY, Activity Assay, Concentration Assay, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Infection

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Polymerase Chain Reaction:

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

RNA Expression:

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

Quantitative RT-PCR:

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

Purification:

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Article Snippet: .. Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US). .. Comparison of a panel of 12 housekeeping genes (GeNorm kit, Primer Design Ltd. UK) identified GAPDH as a stably expressed reference gene.

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    PrimerDesign Inc hcv rna expression
    REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. <t>HCV</t> A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV <t>RNA</t> replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)
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    REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)

    Article Snippet: Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Activation Assay, Expressing, Microarray, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Inhibition, MANN-WHITNEY

    Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

    Article Snippet: Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Expressing, Over Expression

    HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

    Article Snippet: Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

    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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: 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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

    Article Snippet: Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Transduction, Expressing, MANN-WHITNEY, Activity Assay, Concentration Assay, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Infection

    REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d REV-ERBs bind SCD1 promoter. Analyses of available ChIP-seq data in mouse livers 30 demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d REV-ERBs bind SCD1 promoter. Analyses of available ChIP-seq data in mouse livers 30 demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)

    Article Snippet: Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Activation Assay, Expressing, Microarray, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Inhibition, MANN-WHITNEY

    Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

    Article Snippet: Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Expressing, Over Expression

    HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

    Article Snippet: Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

    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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

    Journal: Nature Communications

    Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

    doi: 10.1038/s41467-019-08299-7

    Figure Lengend Snippet: 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 sh Rev-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. sh Rev-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 (IC 50 ) (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% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

    Article Snippet: Real-time reverse transcription PCR Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).

    Techniques: Transduction, Expressing, MANN-WHITNEY, Activity Assay, Concentration Assay, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Infection