key: cord-0918090-qbya7kpo
authors: Zhuang, Xiaodong; Tsukuda, Senko; Wrensch, Florian; Wing, Peter AC.; Schilling, Mirjam; Harris, James M.; Borrmann, Helene; Morgan, Sophie B.; Cane, Jennifer L.; Mailly, Laurent; Thakur, Nazia; Conceicao, Carina; Sanghani, Harshmeena; Heydmann, Laura; Bach, Charlotte; Ashton, Anna; Walsh, Steven; Tan, Tiong Kit; Lisa Schimanski,; A Huang, Kuan-Ying; Schuster, Catherine; Watashi, Koichi; Hinks, Timothy SC.; Jagannath, Aarti; Vausdevan, Sridhar R.; Bailey, Dalan; Baumert, Thomas F.; McKeating, Jane A.
title: The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells
date: 2021-09-16
journal: iScience
DOI: 10.1016/j.isci.2021.103144
sha: b62a4a3a9f16489dc383f60b089a5144756e4d7c
doc_id: 918090
cord_uid: qbya7kpo

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract via Spike glycoprotein binding to angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism’s response to its environment and can regulate host susceptibility to virus infection. We demonstrate that silencing the circadian regulator Bmal1 or treating lung epithelial cells with the REV-ERB agonist SR9009 reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication. Importantly, treating infected cells with SR9009 limits SARS-CoV-2 replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced interferon stimulated gene transcripts in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to limit SARS-CoV-2 infection. Our study highlights alternative approaches to understand and improve therapeutic targeting of SARS-CoV-2.

SR9009 reduced BMAL1 promoter activity and protein expression, with no effect on cell viability ( Figure S3) . 36 SR9009 treatment reduced ACE2 in a dose-dependent manner but had no effect on TMPRSS2 expression ( Figure  37 1D). Furthermore, SARS-CoV-2pp infection was significantly reduced by SR9009 treatment in parental Calu-3 cells 38 but not in shBMAL1 silenced cells, demonstrating a BMAL1-dependency ( Figure 1E ). In contrast, SR9009 treatment 39 had no effect on VSV-Gpp infection ( Figure 1E we showed ISG induction in BMAL1 silenced Calu-3 cells and this was not limited to ISGs with direct antiviral activity 17 but includes those involved in many cellular pathways ( Figure 4E) A key finding from our study is the potential application of chronomodifying drugs for the treatment of SARS-CoV-5 2 infection (Sengupta et al., 2021) . The Bmal1 expression plasmid was previously described (Yang et al., 2020) and further engineered with a Flag tag. 19

The Clock expression plasmid was previously described (Schoenhard et al., 2003) . (Table S1 ) 34 35 RT-qPCR. Cells were washed in PBS then lysed using Tri-reagent (Sigma), and mRNA extracted by phase separation 36 or RNeasy kit (Qiagen). Equal amounts of cDNA were synthesised using the High Capacity cDNA Kit (Applied 37 Biosystems) and mRNA expression determined using Fast SYBR master mix in a StepOne thermocycler (Applied 38

Biosystems) using the ΔΔCt method. The lung tissues were lysed in TRI Reagent (Sigma) using a gentleMACS 39 dissociator and RNA extracted using the manufacturers protocol. cDNAs were synthesized using the Maxima H 40

Minus cDNA Synthesis Kit (Thermo Scientific) and mRNA quantified using iTaq Universal SYBR Green Supermix 41 (Biorad) and specific primers on a QuantStudio 3 RT-PCR system (Applied Biosystems).

Immunoblotting. Cell lysates were prepared by washing cells with PBS, then lysed in Igepal lysis buffer (10mM Tris 1 pH 7.5, 0.25M NaCl, 0.5% Igepal) supplemented with protease inhibitor cocktail (Roche Complete™) at 4 o C for 5 2 min, followed by clarification by centrifugation (3 min, 12,000 rpm). Supernatant was mixed with Laemmli sample 3 buffer, separated by SDS-PAGE and proteins transferred to polyvinylidene difluoride membrane (Immobilon-P, 4 Millipore). Membranes were blocked in 5% milk in PBS/0.1% Tween-20, then incubated with anti-ACE2 (Abcam 5 ab108252), anti-TMPRSS2 (SCBT sc-515727), anti-BMAL1 (Abcam Ab93806) or Anti-β-actin (Sigma A5441) primary 6 antibodies and appropriate HRP-conjugated secondary antibodies (DAKO). Chemiluminescence substrate (West 7 Dura, 34076, Thermo Fisher Scientific) was used to visualize proteins using a ChemiDoc XRS+ imaging system 8 (BioRad). Anti-β-actin-HRP conjugate (Abcam ab49900) and/or Coomassie brilliant blue staining was used to verify 9 equal protein loading and densitometric analysis performed using ImageJ software (NIH). Chromatin extracts from Calu-3 cells were immunoprecipitated using antibodies specific for BMAL1, REV-ERBα or rabbit IgG. qRT-PCR for ACE2 promoter DNA using primers over defined Ebox and RORE motifs or control host gene Per1 for BMAL1 ChIP and Bmal1 for REV-ERBα (positive control) was performed. IP data is presented relative to the rabbit IgG control shown as the dotted line (mean ± S.E.M., n = 2, Kruskal-Wallis ANOVA with Dunn's test). (Related to Fig.1 (A) Differentially expressed genes in SR9009 treated Calu3 cells. Calu-3 cells were treated with SR9009 (20 μM) for 24h and gene expression quantified by RNA sequencing. Differential expression analysis was performed using DESeq2 Package between SR9009 and untreated cells. Volcano plot shows significantly differentially expressed genes based on a log2FC of +/-1 and an adjusted (Benjamini Hochberg) P value of 0.05. Red points denote significant upregulation, blue denotes downregulation. (B) Validation of RNAseq data on previously reported clock genes. All Gene sets from MSigDB were searched for the term "Circadian", and 24 gene sets obtained. GSEA was performed between shBmal1 and SR9009 against untreated cells. Gene sets were filtered for size based on >15 and <500 genes, and for their expression in both experimental groups. 9/12 circadian gene sets were significantly enriched in shBmal1, and 4/13 SR9009 treated cells. Gene sets are ranked by NES and coloured by FDR. (C) Gene set enrichment analysis (GSEA) for SR9009 regulated host pathways. Using the Hallmarks gene sets from the molecular signatures database, 18 out of 50 gene sets were significantly upregulated in SR9009 above untreated cells, at an FDR of less than 25%. Significantly enriched hallmarks were plotted, ranked by normalised enrichment score, and coloured by FDR. (Related to Fig.4) . 

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation

Patient fibroblast circadian rhythms predict lithium 5 sensitivity in bipolar disorder

Circadian Rhythms in the Pathogenesis and Treatment of Fatty Liver

Clocking in to immunity

Adrenergic nerves govern circadian leukocyte recruitment to tissues

Circadian Control of DRP1 Activity Regulates Mitochondrial Dynamics and 15

PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2

Clocks

Lessons for the COVID-19 Pandemic

Reciprocal Regulation between the Circadian Clock and Hypoxia Signaling at the Genome Level in Mammals

Pathological 4 findings of COVID-19 associated with acute respiratory distress syndrome. The Lancet

COVID-19 patients and its underlying treatment options

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19

Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion

Single-Cell RNA Expression Profiling of ACE2, the 19 Receptor of SARS-CoV-2

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent 22 patient

The circadian clock components BMAL1 and REV-ERBalpha regulate flavivirus replication. 25 Nature communications 10

RRID:AB_10864415 Mouse anti-TMPRSS2

Bacterial and virus strains SARS-CoV-2 Victoria 01

SARS-CoV-2 B1.1.7: 20I/501Y.V1.HMPP1 (Tegally et al., 2020) Public Health England SARS-CoV-2 B1

2021) Centre for the AIDS Programme of Research in South Africa