cloning dyrk1a  (Addgene inc)

Journal: bioRxiv

Article Title: Kinase-independent activity of DYRK1A promotes viral entry of highly pathogenic human coronaviruses

doi: 10.1101/2022.09.13.507833

Figure Lengend Snippet: (A) XY plot comparing the top 10,000 enriched genes that promote SARS-CoV-2 infection in genome-wide CRISPR screens performed in Wei et al . 2021 (Vero-E6 cells, African green monkey kidney cells) and Biering et al . 2022 (Calu-3 cells, human lung epithelial cells). DYRK1A scored as the most strongly enriched gene after ACE2, supporting a conserved pro-viral role for DYRK1A in monkey and human cells. (B) Immunoblot for two single-cell monoclonal knockouts (KO) of DYRK1A (KO#1 and KO#2) generated from parental Vero-E6 cells. Cells lacking DYRK1A were confirmed by Sanger sequencing. (C) Wild-ype (WT) Vero-E6 cells and DYRK1A KO cells were infected with icSARS-CoV-2-mNeonGreen (mNG) at an MOI ~ 1.0 and imaged at 48 hours post-infection (hpi) (left). mNeonGreen+ expressing cell frequency (%mNG) was quantified from stitched images (right). Scale bar: 1000 μm. (D) DYRK1A KO and WT Vero-E6 cells were infected with SARS-CoV-2, HKU5-SARS-CoV-S, or MERS-CoV at an MOI ~ 1.0 and cell viability was assessed at 72 hpi with CellTiter Glo. % Viability was calculated relative to uninfected controls. (E) DYRK1A KO and WT Vero-E6 cells were infected with VSV pseudovirus (VSVpp) encoding CoV spike proteins and a Renilla luciferase (Rluc) reporter at 24 hpi. % Entry for VSVpp-Rluc-SARS2-S, VSVpp-Rluc-SARS-S, and VSVpp-Rluc-MERS-S was normalized to VSVpp-Rluc-VSV-G control and WT Vero-E6 cells. Data were analyzed by unpaired Student’s t-test; * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.001. Shown are means ± SEM. Data in (C) , (D) , and (E) are representative of three independent biological experiments performed with at least 3 technical replicates.

Article Snippet: For lentiviral transduction, Vero-E6 or Calu-3 cells were transduced at 50% confluency and selected with puromycin 48 hours later. hACE2 and hDPP4 overexpressing lines were generated by stable lentiviral delivery of pLV-EF1a-ACE2-puro (gift of A. Iwasaki) and pLEX307-DPP4-puro (Addgene #158451) into DYRK1A knockout clones.

Techniques: Infection, Genome Wide, CRISPR, Western Blot, Generated, Sequencing, Expressing, Luciferase, Control

Journal: bioRxiv

Article Title: Kinase-independent activity of DYRK1A promotes viral entry of highly pathogenic human coronaviruses

doi: 10.1101/2022.09.13.507833

Figure Lengend Snippet: (A) Immunoblot for ACE2 in WT Vero-E6 and DYRK1A KO cells. (B) mRNA abundance of ACE2 and DPP4 transcripts in WT Vero-E6 and DYRK1A KO clones assessed by qPCR. ddCT values are calculated relative to actin and normalized to WT Vero-E6 values. (C) WT Vero-E6, DYRK1A KO#1, and DYRK1A KO#1 overexpressing recombinant human ACE2 (hACE2) were infected with VSVpp-Rluc-SARS2-S and % entry was assessed at 24 hpi. Immunoblot confirms rescue of ACE2 expression in DYRK1A KO#1. (D) WT Vero-E6, DYRK1A KO#2, and DYRK1A KO#2 overexpressing recombinant human DPP4 (hDPP4) were infected with VSVpp-Rluc-MERS-S and % entry was assessed at 24 hpi. Immunoblot confirms DPP4 overexpression in DYRK1A KO#2. % Entry in (C) and (D) was normalized to VSVpp-Rluc-VSV-G and WT Vero-E6 cells. Data were analyzed by unpaired Student’s t-test; ns p> 0.05, * p< 0.05, ** p< 0.01, *** p< 0.001. Shown are means ± SEM. Each datapoint is the mean of 3-5 technical replicates. All experiments were performed in biological replicate.

Article Snippet: For lentiviral transduction, Vero-E6 or Calu-3 cells were transduced at 50% confluency and selected with puromycin 48 hours later. hACE2 and hDPP4 overexpressing lines were generated by stable lentiviral delivery of pLV-EF1a-ACE2-puro (gift of A. Iwasaki) and pLEX307-DPP4-puro (Addgene #158451) into DYRK1A knockout clones.

Techniques: Western Blot, Clone Assay, Recombinant, Infection, Expressing, Over Expression

Journal: bioRxiv

Article Title: Kinase-independent activity of DYRK1A promotes viral entry of highly pathogenic human coronaviruses

doi: 10.1101/2022.09.13.507833

Figure Lengend Snippet: (A) DYRK1A protein domains and engineered point mutations. Nuclear localization mutants were generated by deletion of the bipartite nuclear localization motif and addition of a C-terminal nuclear export signal. (B) Immunoblot for cells expressing an empty vector (KO+vec), WT DYRK1A (KO+WT), kinase dead DYRK1A (KO+K188R and KO+Y321F), or a nuclear localization mutant DYRK1A (KO+NES) were reintroduced to DYRK1A KO#1. Constructs were tagged with 3X FLAG and induced by doxycycline (DOX) for 72 hours. Reintroduction of DYRK1A rescues ACE2 expression was rescued relative to WT Vero-E6 cells. (C) Immunoblot after cytosolic-nuclear fractionation of DYRK1A KO+WT or DYRK1A KO+NES, demonstrating that DYRK1A is predominantly localized to the nucleus until disruption of the bipartite nuclear localization motif and addition of a nuclear export signal. DYRK1A expression was induced by DOX for 72 hours prior to fractionation. (D) WT Vero-E6 cells and cells overexpressing DYRK1A after DOX induction for 72 hours were infected with icSARS-CoV-2-mNeonGreen (mNG) at MOI ~ 1. Cells were imaged and mNeonGreen+ expressing cell frequency (%mNG) was quantified from stitched images. (E) WT Vero-E6 cells and DYRK1A KO cells with reintroduced DYRK1A were infected with VSVpp-Rluc-SARS2-S or VSVpp-Rluc-MERS-S and % entry was assessed at 24 hpi. % Entry was normalized to VSVpp-Rluc-VSV-G and WT Vero-E6 cells. (F) 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 ~ 1) and cell viability was assessed 72 hpi via CellTiter Glo. % Viability was calculated relative to uninfected or untreated controls. Data were analyzed by Kruskal-Wallis (C, left), ordinary one-way ANOVA (D, E left), or two-way ANOVA multiple comparisons tests (E right); ns p> 0.05, * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001. Data shown are means ± SEM of (D, E) 5-10 technical replicates across three independent experiments and (F) 2-3 technical replicates performed in biological duplicate.

Article Snippet: For lentiviral transduction, Vero-E6 or Calu-3 cells were transduced at 50% confluency and selected with puromycin 48 hours later. hACE2 and hDPP4 overexpressing lines were generated by stable lentiviral delivery of pLV-EF1a-ACE2-puro (gift of A. Iwasaki) and pLEX307-DPP4-puro (Addgene #158451) into DYRK1A knockout clones.

Techniques: Generated, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Construct, Fractionation, Disruption, Infection, Positive Control, Protease Inhibitor

Journal: bioRxiv

Article Title: Kinase-independent activity of DYRK1A promotes viral entry of highly pathogenic human coronaviruses

doi: 10.1101/2022.09.13.507833

Figure Lengend Snippet: (A) Principal component analysis of RNA-Seq experiments performed in WT Vero-E6, KO+vec, KO+WT, and KO+Y321F cells. Rep refers to independent biological replicates. (B) Heatmap depicting differentially expressed genes by RNA-Seq in WT Vero-E6, KO+vec, KO+WT, and KO+Y321F cells. (C) XY plot of RNA-Seq L2FC versus CRISPR z-scores in Vero-E6 cells ( Wei et al . 2022 ) for SARS-CoV-2 (left), MERS-CoV (middle), or VSV-SARS-CoV-2-S (right). Denoted are receptor ( ACE2, DPP4 ) and protease ( CTSL ) genes as significantly upregulated pro-viral genes of interest. (D) qPCR for ACE2 validates RNA-Seq results and supports partial rescue of ACE2 mRNA transcripts in cells where DYRK1A-WT or DYRK1A-Y321F are reintroduced. (E) Principal component analysis of ATAC-Seq experiments performed in WT Vero-E6, KO+vec, KO+WT, and KO+Y321F cells. Rep refers to independent biological replicates. (F) ATAC-Seq gene tracks for ACE2 , highlighting increased accessibility at putative enhancers and near the transcriptional start site (TSS) in the presence of DYRK1A. (G) ATAC-Seq gene tracks for DPP4 , showing increased chromatin accessibility in the presence of DYRK1A. (H) ATAC-Seq genome tracks for CTSL , showing increased chromatin accessibility in the presence of DYRK1A. All experiments were performed in biological duplicate (RNA-seq/ATAC-seq) or triplicate (qPCR).

Article Snippet: For lentiviral transduction, Vero-E6 or Calu-3 cells were transduced at 50% confluency and selected with puromycin 48 hours later. hACE2 and hDPP4 overexpressing lines were generated by stable lentiviral delivery of pLV-EF1a-ACE2-puro (gift of A. Iwasaki) and pLEX307-DPP4-puro (Addgene #158451) into DYRK1A knockout clones.

Techniques: RNA Sequencing, CRISPR

Journal: bioRxiv

Article Title: Kinase-independent activity of DYRK1A promotes viral entry of highly pathogenic human coronaviruses

doi: 10.1101/2022.09.13.507833

Figure Lengend Snippet: (A) Polyclonal knockouts of DYRK1A were generated in Calu-3 human lung epithelial cells with two independent guides (sgRNA#1 and sgRNA#2). Viral titers were assessed after 24 hpi by TCID 50 and compared against a non-targeting guide (NTG). (B) Conditional deletion and infection schematic for hACE2 KI/het Ubc CreERT2 and DYRK1A F/F Ubc CreERT2. (C) ACE2 was conditionally deleted from a hACE2 knock-in (KI) mouse under control of a tamoxifen inducible Cre recombinase ( hACE2 KI/het Ubc CreERT2). Cre+ and Cre- mice were treated with tamoxifen for five days and were then infected intranasally with 10 5 PFU SARS-CoV-2 (WA1/2020). (D) DYRK1A was conditionally deleted from a DYRK1AF/F mouse ( DYRK1A F/F Ubc CreERT2). Cre+ and Cre- mice were treated with tamoxifen for five days and were then infected intranasally with 10 5 PFU MA10. In (C, D) , lung homogenates containing virus were harvested at 1-day post-infection. Lung viral titers were assessed by plaque assay and are reported as plaque forming units (PFU)/mL. Data shown are means ± SEM from (A) five, (C) three, or (D) two independent experiments. Each experiment in (C, D) included at least three mice per group. Data were analyzed by unpaired Student’s t-test; ** p< 0.01.

Article Snippet: For lentiviral transduction, Vero-E6 or Calu-3 cells were transduced at 50% confluency and selected with puromycin 48 hours later. hACE2 and hDPP4 overexpressing lines were generated by stable lentiviral delivery of pLV-EF1a-ACE2-puro (gift of A. Iwasaki) and pLEX307-DPP4-puro (Addgene #158451) into DYRK1A knockout clones.

Techniques: Generated, Infection, Knock-In, Control, Virus, Plaque Assay