Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2–host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism

The KDM6A-KMT2D-p300 axis regulates susceptibility to diverse coronaviruses by mediating viral receptor expression.

Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses

DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses

DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner.

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses

DYRK1A is coexpressed with ACE2 and DPP4 in human lung epithelial cells.

(A, B) Scatter plots and (C, D) dot plots assessing scaled average expression of DYRK1A and (A-E) DPP4 or (C, D) ACE2 from existing scRNA-seq datasets [94,95]. Data in (A. B) were analyzed by Pearson and Spearman correlation statistical tests. ACE2, angiotensin-converting enzyme 2; DPP4, dipeptidyl peptidase-4; DYRK1A, Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A; scRNA-seq, single-cell RNA sequencing. (PDF)</p

DYRK1A and SMARCA4 share limited similarities in regulating chromatin accessibility and gene expression.

(A) Principal component analysis of ATAC-Seq experiments performed in DYRK1A KO+vec, DYRK1A KO+WT, SMARCA4 KO+vec, and SMARCA4 KO+WT cells generated in a parental Vero-E6 background. Each experiment was performed in biological duplicate (replicates 1 and 2). DYRK1A and SMARCA4 loss share some molecular impacts, suggesting that some pathways may be coregulated (PC1), whereas others may be independently regulated (PC2). (B) Correlation heatmap comparing all sites from ATAC-Seq experiments in DYRK1A KO+vec, DYRK1A KO+WT, SMARCA4 KO+vec, and SMARCA4 KO+WT cells. (C) Correlation heatmap comparing chromatin accessibility by ATAC-Seq in DYRK1A or SMARCA4 complemented cells, identifying a correlation coefficient of 0.33 supporting approximately 33% of clusters may be correlated by DYRK1A and SMARCA4. (D) Venn diagram highlighting shared peaks gained by DYRK1A and SMARCA4 complementation. (E) Correlation heatmap comparing changes in RNA abundance in DYRK1A or SMARCA4 complemented cells, identifying a correlation coefficient of 0.08 supporting (F) Gene set enrichment analysis from RNA-Seq experiments showing shared pathway regulation by DYRK1A and SMARCA4. Data underlying this figure can be found under GEO Accessions GSE213999 and GSE186201. DYRK1A, Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A; KO, knockout; WT, wild-type. (PDF)</p

DYRK1A active site inhibitors do not block SARS-CoV-2 infection at tolerated concentrations.

(A) WT Vero-E6 cells, DYRK1A KO cells, and cells overexpressing DYRK1A after DOX induction for 72 hours were infected with SARS-CoV-2 WA/01/2020 at an MOI of approximately 0.1 for 1, 24, or 48 hours. Plaque assays were performed in biological duplicate. Shown is 1 representative replicate. (B) WT Vero-E6 cells were treated with the positive control protease inhibitor calpain inhibitor III or a potent DYRK1A inhibitor (harmine, INDY, DYR219, and DYR533) for 48 hours. Cells were then infected with SARS-CoV-2 (MOI approximately 1), and cell viability was assessed 72 hpi via CellTiter-Glo. % Viability was calculated relative to uninfected or untreated controls. The half-maximal inhibitory concentration (IC50) and half-maximal cytotoxic concentration (CC50) were calculated for each drug using nonlinear regression dose response curves. Each experiment was performed at least 2 independent times. Shown are the means of 2–3 technical replicates. Data underlying this figure can be found in S1 Data. DOX, doxycycline; DYRK1A, Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A; hpi, hours postinfection; KO, knockout; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type. (PDF)</p

DYRK1A regulates global gene expression and chromatin accessibility.

(A) RNA-Seq volcano plots depicting DEGs in cells where DYRK1A is absent or reintroduced. (B) Enrichr pathway analysis for biological function enriched in the presence of DYRK1A. Gene set criteria included p 1.5 for both KO+WT and KO+Y321F vs. KO+vec. (C) ATAC-Seq volcano plots depicting DEGs in cells where DYRK1A is absent or reintroduced. All experiments were performed in biological duplicate. Data underlying this figure can be found under GEO Accession GSE213999. DEG, differentially expressed gene; DYRK1A, Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A; KO, knockout; L2FC, log2 fold-change; WT, wild-type. (PDF)</p

The regulatory role of DYRK1A on Ace2 expression is not conserved in mice.

DYRK1A was conditionally deleted by treating Dyrk1aAF/F Ubc CreERT2 mice with tamoxifen for 5 consecutive days. On day 6, (A) lung and (B) distal small intestine homogenates were assessed by RT-qPCR for Ace2 expression. Each data point represents an individual mouse. Error bars represent ± SEM, and statistical comparisons were generated via Student t test; ns p > 0.05. Each experiment was performed 2 independent times with at least 3 mice per group. Shown the ΔΔCT values for each mouse normalized to actin and Cre negative controls. Data underlying this figure can be found in S1 Data. ACE2, angiotensin-converting enzyme 2; DYRK1A, Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A; RT-qPCR, quantitative reverse transcription PCR. (PDF)</p