DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner
Madison S. Strine, Wesley L. Cai, Jin Wei, Mia Madel Alfajaro, Renata B. Filler, Scott B. Biering, Sylvia Sarnik,Ryan D. Chow, Ajinkya Patil, Kasey S. Cervantes, Clayton K. Collings, Peter C. DeWeirdt, Ruth E. Hanna, Kevin Schofield, Christopher Hulme, Silvana Konermann, John G. Doench, Patrick D. Hsu, Cigall Kadoch, Qin Yan, Craig B. Wilen
Abstract
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.
Introduction
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), is a beta coronavirus that has launched an ongoing pandemic that continues to threaten public health globally [1,2]. Two additional beta coronavirus family members (SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV)) have caused more limited epidemics but with a higher case fatality rate [3–5]. There are also 4 endemic alpha and beta human coronaviruses that cause the common cold (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1). Despite zoonotic outbreaks of 3 beta coronaviruses in less than 20 years, our understanding of host factors that support these highly pathogenic human coronaviruses and the immune response to them remain incompletely understood [1,6,7].
Material and methods
Cell culture
HEK293T (ATCC) and Vero-E6 (ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) with 5% heat-inactivated fetal bovine serum (FBS, VWR) and 1% Penicillin/Streptomycin (Gibco) unless otherwise noted. Vero-E6-ACE2-TMPRSS2 (gift from Barney Graham, NIH) were cultured with 5% DMEM, 5% FBS, 1% Penicillin/Streptomycin, and 5 μg/ml puromycin (Gibco). Calu-3 (ATCC) cells were cultured in RPMI 1640 (Gibco) with 1% Glutamax 100X (Gibco), 10% FBS, 1% Penicillin/Streptomycin, and 16 ng/ml hepatocyte growth factor (HGF, Stem Cell Technologies). When selecting cells transduced by lentivirus, Vero-E6 cells and Calu-3 were treated with 5 μg/ml or 1 μg/ml puromycin (Gibco), respectively. All cells were grown at 37°C in 5% CO2.
Results
DYRK1A promotes viral entry for SARS-CoV-2, SARS-CoV, and MERS-CoV
We and others recently identified DYRK1A as a critical host factor for SARS-CoV-2, MERS-CoV, and chimeric HKU5 (bat coronavirus) expressing the SARS-CoV spike (HKU5-SARS-CoV-S) in a genome-wide CRISPR/Cas9 inactivation screen in Vero-E6 cells [23,34]. In 2 additional independent genome-wide CRISPR screens in Calu-3 human immortalized lung cancer cells, DYRK1A was also identified as a top proviral gene for SARS-CoV-2 [24,34]. By comparing the top 10,000 enriched genes ranked by z-score for SARS-CoV-2 across Vero-E6 and Calu-3 cells, we identified DYRK1A as second most strongly enriched gene after only ACE2 [23,24] (Fig 1A). In a separate screen, DYRK1A was the third most strongly enriched hit after ACE2 [34]. We generated single-cell knockout (KO) clones of DYRK1A in Vero-E6 cells using CRISPR/Cas9 to validate screening results and clarify the proviral mechanism underlying DYRK1A activity in coronavirus infection (Fig 1B).
Discussion
A number of genome-wide CRISPR/Cas9 screens have been performed to unveil host factors that regulate SARS-CoV-2 infection [23,24,28–34,98]. We previously identified DYRK1A as a proviral gene for both SARS-CoVs and MERS-CoV in Vero-E6 cells and Calu-3 cells [23,24]. Additional recent independent screens in Vero-E6 and Calu-3 cells have confirmed our initial finding of DYRK1A as a host dependency factor for SARS-CoV-2 [34,35]. Numerous other SARS-CoV-2 genome-wide CRISPR KO screens failed to identify DYRK1A—a disparity likely attributable to their reliance on cells that ectopically overexpress ACE2. In such cells (A549-ACE2 and Huh7.5-ACE2), ACE2 regulation is uncoupled from the transcriptional regulators that promote endogenous ACE2 expression, rendering them nonessential for coronavirus infection [28–31]. As a result, numerous transcription factors and epigenetic regulators are obscured in screening results.
Acknowledgments
We would like to acknowledge the Yale Center for Molecular Discovery, Yale Center for Genome Analysis, and the Yale Flow Cytometry Facility for technical assistance and sample processing, Nathan Grubaugh for sequence validation of viral stocks, and BEI Resources, the World Reference Center for Emerging Viruses and Arboviruses (WRECVA), Moitrayee Bhattacharyya, Akiko Iwasaki, Katerina Politi, Yoshihiko Miyata, Man Mohan, and Vance Lemmon for critical reagents and expertise.
Citation: Strine MS, Cai WL, Wei J, Alfajaro MM, Filler RB, Biering SB, et al. (2023) DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner. PLoS Biol 21(6): e3002097. https://doi.org/10.1371/journal.pbio.3002097
Academic Editor: Frank Kirchhoff, Ulm University Medical Center, GERMANY
Received: October 26, 2022; Accepted: March 29, 2023; Published: June 13, 2023
Copyright: © 2023 Strine et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are included in the article or supplement, including source data (S1 Data). The RNA-Seq and ATAC-Seq for DYRK1A are available at NCBI GEO under accession GSE213999. The analysis code generated during the study for DYRK1A RNA-Seq and ATAC-Seq are available at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213999. SMARCA4 RNA-Seq and ATAC-Seq are available at GEO under accession GSE186201 at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE186201. Human single-cell RNA-Seq datasets are available under the Human Cell Atlas Data Coordination Platform and NCBI BIOPROJECT Accession code PRJEB31843 at: https://www.ncbi.nlm.nih.gov/bioproject/PRJEB31843 and the European Genome-phenome Archive under accession EGAS00001004344 at: https://ega-archive.org/studies/EGAS00001004344. All mice are available for purchase at the Jackson Laboratories. Viral stocks and plasmids are available BEI or Addgene or can be made available under Material Transfer Agreement. For initiation of the Material Transfer Agreement process, contact the corresponding author (Craig B. Wilen, craig.wilen@yale.edu)
Funding: This work was supported by the Burroughs Wellcome Fund (C.B.W.); the Robert E. Leet and Clara Guthrie Patterson Trust (C.B.W.); the Smith Family Foundation (C.B.W.); National Science Foundation DGE1752134 (M.S.S.); and NIH grants K08 A1128043 (C.B.W.), R01 AI148467 (C.B.W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: C.K. is the scientific founder, Scientific Advisor to the Board of Directors, Scientific Advisory Board member, shareholder, and consultant for Foghorn Therapeutics. C.K. also serves on the Scientific Advisory Boards of Nereid Therapeutics (shareholder and consultant), Nested Therapeutics (shareholder and consultant) and Fibrogen (consultant) and is a consultant for Cell Signaling Technologies and Google Ventures (shareholder and consultant). C.B.W. is a consultant for Exscientia. The other authors declare no competing interests.
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002097#abstract0
