Journal: Journal of Virology

Article Title: DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner

doi: 10.1128/jvi.01239-23

Figure Lengend Snippet: DYRK1A, but not DYRK1B, is a host factor essential for TGEV replication. (A) Western blot analysis validated the protein expression level of endogenous DYRK1A in DYRK1A KO cells and WT cells. GAPDH was used as an internal control gene. (B) Western blot assay to detect the TGEV N protein expressed in DYRK1A KO and WT cells following infection with TGEV [multiplicity of infection (MOI) = 0.1], at 12 and 24 hours post-infection (hpi). GAPDH was used as an internal control gene. (C) Multiple step viral growth assay. The viral titers in DYRK1A KO and WT cells infected with TGEV (MOI = 0.1) were monitored at different time points (12, 24, 36, and 48 h). (D and E) DYRK1A KO and WT cells were infected with TGEV at different MOIs (0.01, 0.1, and 1). Immunofluorescence assays (D) were used to detect the TGEV N protein expression following infection with TGEV at 24 hpi. Scale bar, 400 µm. TGEV titers (E) were tested at 24 hpi. (F) Rescue assays for WT, DYRK1A KO, and DYRK1A KO-rescue cells infected with TGEV (MOI = 0.1) at 24 hpi. The TGEV N copy number was assessed by quantitative real-time PCR. (G) Overexpression of DYRK1A in PK-15 control cells following infection with TGEV (MOI = 0.01) at 24 hpi. RT-qPCR assay for determination of the absolute mRNA level of the TGEV N gene. WT, transfection of the pcDNA3.1 empty vector in PK-15 cells; WT-overexpression, transfection of the pcDNA3.1-DYRK1A vector in PK-15 cells. (H and I) DYRK1B KO and WT cells were infected with TGEV (MOI = 0.01). Immunofluorescence assays (H) were used to detect TGEV N protein expression following infection with TGEV at 24 hpi. Scale bar, 400 µm. TGEV titers (I) were tested at 24 hpi. (J) PK-15 cells were incubated in advance with the DYRK1B inhibitor AZ191 at different concentrations (12.5, 6.25, 3.125, and 1.5625 µM) for 2 h, and the cells treated with the inhibitor were infected with TGEV (MOI = 0.01). The TGEV titers were tested at 24 hpi. The means and SDs of the results from three independent experiments are shown. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. P-values were determined by two-tailed unpaired Student’s t-tests.

Article Snippet: The subcellular localization of DYRK1A in DYRK1A KO cells and control cells transfected with DYRK1A-FLAG-pcDNA3.1 plasmid was observed after 24 h. Afterward, TGEV (MOI = 1) was added into these cells, incubated for 24 h, immunolabeled with a FLAG-tag antibody (Proteintech, no. 20543-1-AP; MBL, no. PM020) and dsRNA antibody (SCICONS, no. 10010200, 1:1,000), and imaged to identify double-fluorescent positive cells. . Statistical analysis Statistical significance values were assessed using GraphPad Prism 8.0.

Techniques: Western Blot, Expressing, Control, Infection, Growth Assay, Immunofluorescence, Real-time Polymerase Chain Reaction, Over Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Incubation, Two Tailed Test

Journal: Journal of Virology

Article Title: DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner

doi: 10.1128/jvi.01239-23

Figure Lengend Snippet: DYRK1A regulates TGEV replication independent of its kinase activity. (A) Western blot analysis validated the protein expression level of DYRK1A in WT, DYRK1A KO, DYRK1A KO-rescue, and DYRK1A KO-rescue-K188R cells. GAPDH was used as an internal control gene. (B) Rescue assays for WT, DYRK1A KO, DYRK1A KO-rescue, and DYRK1A KO-rescue-K188R cells infected with TGEV (MOI = 0.01). Viral titers were tested at 24 hpi. (C and E) PK-15 cells were treated with or without INDY (C) and Harmine (E) for 48 h, and cell viability was measured by the MTS assay. (D) PK-15 cells were incubated in advance with different concentrations (25, 12.5, 6.25, 3.125, and 1.5625 µM) of INDY for 2 h, and cells treated with the inhibitor were infected with TGEV (MOI = 0.01). The TGEV titers were tested at 24 hpi. (F and G) PK-15 cells were incubated in advance with different concentrations (12.5, 6.25, 3.125, and 1.5625 µM) of Harmine for 2 h, and cells treated with the inhibitor were infected with TGEV (MOI = 0.01). The TGEV titers (F) were tested at 24 hpi. The TGEV N protein was detected by the immunofluorescence assay (G). Scale bar, 400 µm. The means and SD of the results from three independent experiments are shown. *P < 0.05; **P < 0.01; ****P < 0.0001. P-values were determined by two-tailed unpaired Student’s t-tests.

Article Snippet: The subcellular localization of DYRK1A in DYRK1A KO cells and control cells transfected with DYRK1A-FLAG-pcDNA3.1 plasmid was observed after 24 h. Afterward, TGEV (MOI = 1) was added into these cells, incubated for 24 h, immunolabeled with a FLAG-tag antibody (Proteintech, no. 20543-1-AP; MBL, no. PM020) and dsRNA antibody (SCICONS, no. 10010200, 1:1,000), and imaged to identify double-fluorescent positive cells. . Statistical analysis Statistical significance values were assessed using GraphPad Prism 8.0.

Techniques: Activity Assay, Western Blot, Expressing, Control, Infection, MTS Assay, Incubation, Immunofluorescence, Two Tailed Test

Journal: Journal of Virology

Article Title: DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner

doi: 10.1128/jvi.01239-23

Figure Lengend Snippet: Nucleus-located DYRK1A facilitates TGEV replication. (A) Confocal fluorescence microscopy analysis of the subcellular localization of TGEV N (indicated in green) and ectopic expression of DYRK1A-FLAG (indicated in red) in WT cells mock-infected (above) or infected with TGEV (MOI = 1), at 12 hpi (middle) and 24 hpi (bottom). Scale bar, 10 µm. (B) Schematic representation of the construction of full-length DYRK1A (above) and DYRK1A-ΔNLS mutant (DYRK1A with deletion of the nuclear localization motif) (bottom). (C) Assessment of the subcellular location of ectopically expressed full-length DYRK1A (above) and DYRK1A-ΔNLS (bottom) (indicated in green) in WT cells via confocal fluorescence microscopy. Scale bar, 5 µm. (D) Overexpression of full-length DYRK1A and DYRK1A-ΔNLS in PK-15 control cells following infection with TGEV (MOI = 0.01). TGEV titers were tested at 24 hpi. The means and SD of the results from three independent experiments are shown. *P < 0.05; **P < 0.01. P-values were determined by two-tailed unpaired Student’s t-tests.

Article Snippet: The subcellular localization of DYRK1A in DYRK1A KO cells and control cells transfected with DYRK1A-FLAG-pcDNA3.1 plasmid was observed after 24 h. Afterward, TGEV (MOI = 1) was added into these cells, incubated for 24 h, immunolabeled with a FLAG-tag antibody (Proteintech, no. 20543-1-AP; MBL, no. PM020) and dsRNA antibody (SCICONS, no. 10010200, 1:1,000), and imaged to identify double-fluorescent positive cells. . Statistical analysis Statistical significance values were assessed using GraphPad Prism 8.0.

Techniques: Fluorescence, Microscopy, Expressing, Infection, Mutagenesis, Over Expression, Control, Two Tailed Test

Journal: Journal of Virology

Article Title: DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner

doi: 10.1128/jvi.01239-23

Figure Lengend Snippet: Knockout of DYRK1A inhibits TGEV entry by downregulating the expression of ANPEP. (A) DYRK1A KO and WT cells were infected with TGEV (MOI = 50) at 4°C for 1 h and assessed for TGEV adsorption. The cells were harvested, and viral RNA was extracted to determine virion attachment at the cell surface. (B) DYRK1A KO and WT cells were infected with TGEV (MOI = 50) at 4°C for 1 h, followed by incubation at 37°C for 30 min. The internalization of TGEV in DYRK1A KO and WT cells was evaluated by absolute quantitative real-time PCR. (C) Membrane fusion assay assessed the fusion ability of the TGEV spike protein in WT cells and DYRK1A KO cells. (D) Volcano plot of differentially expressed genes in WT and DYRK1A KO PK-15 cells. The expression of ANPEP, ACE2, and DPP4 was strongly reduced in the absence of DYRK1A. (E) RT-qPCR assay for determination of relative mRNA levels of ANPEP in WT, DYRK1A KO, DYRK1A KO-rescue, and DYRK1A KO-rescue-K188R cells. (F) RT-qPCR validation of mRNA expression of endocytic pathway genes enriched according to RNA-seq. (G) RT-qPCR assay for determination of relative mRNA levels of ANPEP in WT, DYRK1A KO, and ANPEP-overexpressing DYRK1A KO cells. (H) WT, DYRK1A KO, and ANPEP-overexpressing DYRK1A KO cells were infected with TGEV (MOI = 50) at 4°C for 1 h and assessed for TGEV adsorption by RT-qPCR assay. (I) WT, DYRK1A KO, and ANPEP-overexpressing DYRK1A KO cells were infected with TGEV (MOI = 50) at 4°C for 1 h, followed by incubation at 37°C for 30 min. The internalization of TGEV was evaluated by absolute quantitative real-time PCR. (J) WT, DYRK1A KO, and ANPEP-overexpressing DYRK1A KO cells were infected with TGEV (MOI = 0.01). Viral titers were tested at 24 hpi. The means and SD of the results from three independent experiments are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P-values were determined by two-tailed unpaired Student’s t-tests.

Article Snippet: The subcellular localization of DYRK1A in DYRK1A KO cells and control cells transfected with DYRK1A-FLAG-pcDNA3.1 plasmid was observed after 24 h. Afterward, TGEV (MOI = 1) was added into these cells, incubated for 24 h, immunolabeled with a FLAG-tag antibody (Proteintech, no. 20543-1-AP; MBL, no. PM020) and dsRNA antibody (SCICONS, no. 10010200, 1:1,000), and imaged to identify double-fluorescent positive cells. . Statistical analysis Statistical significance values were assessed using GraphPad Prism 8.0.

Techniques: Knock-Out, Expressing, Infection, Adsorption, Incubation, Real-time Polymerase Chain Reaction, Membrane, Single Vesicle Fusion Assay, Quantitative RT-PCR, Biomarker Discovery, RNA Sequencing, Two Tailed Test

Journal: Journal of Virology

Article Title: DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner

doi: 10.1128/jvi.01239-23

Figure Lengend Snippet: DYRK1A KO inhibits viral RNA synthesis of TGEV by suppressing DMV formation. (A) Evaluation of the effects of DYRK1A KO on virus particle assembly by TEM experiment. Compared with numerous virions (yellow arrows) found in WT cells, few virus-like particles wrapped in vesicles of varying sizes were found in DYRK1A KO cells. Scale bars, 1 µm or 500 nm. Mock, uninfected cells; M, mitochondria; ER, endoplasmic reticulum; N, nucleus. (B) Assessment of the TGEV release stage in DYRK1A KO and WT cells infected with TGEV (MOI = 0.1). Intracellular and extracellular viral titers were evaluated by virus median tissue culture infectious dose (TCID50) assays at 24 hpi. (C) Confocal microscopy to evaluate early-stage TGEV replication by detecting dsRNA formation in WT and DYRK1A KO cells infected with TGEV (MOI = 5) at 3 hpi. Scale bars, 10 µm. (D) DYRK1A KO and WT cells were infected with TGEV at an MOI of 0.01. Positive (+vRNA) or negative (−vRNA) viral RNA was quantified by RT-qPCR. (E) Infectious TGEV-BAC plasmids were transfected into WT, DYRK1A KO, DYRK1A KO-rescue, and DYRK1A KO-rescue-K188R cells. After 8 h, they were incubated with an anti-TGEV S protein antibody, and after 48 h, the cells were collected and subjected to RT-qPCR. (F) Evaluation of DMV formation in WT, DYRK1A KO, ANPEP-overexpressing DYRK1A KO, DYRK1A KO-rescue, and DYRK1A KO-rescue-K188R cells by transmission electron microscopy. Numerous typical DMVs (diameter 150–300 nm) were observed in TGEV-infected PK-15 cells, DYRK1A KO-rescue cells, and DYRK1A KO-rescue-K188R cells (MOI = 1, 12 hpi) (red arrows). TGEV-infected DYRK1A KO and ANPEP-overexpressing DYRK1A KO cells contained several monolayer vesicles. Scale bar, 1 µm or 500 nm as indicated. (G) Quantification of DMV numbers per cell is shown as the mean ± SD (n = 20). The means and SD of the results from three independent experiments are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P-values were determined by two-tailed unpaired Student’s t-tests.

Article Snippet: The subcellular localization of DYRK1A in DYRK1A KO cells and control cells transfected with DYRK1A-FLAG-pcDNA3.1 plasmid was observed after 24 h. Afterward, TGEV (MOI = 1) was added into these cells, incubated for 24 h, immunolabeled with a FLAG-tag antibody (Proteintech, no. 20543-1-AP; MBL, no. PM020) and dsRNA antibody (SCICONS, no. 10010200, 1:1,000), and imaged to identify double-fluorescent positive cells. . Statistical analysis Statistical significance values were assessed using GraphPad Prism 8.0.

Techniques: Virus, Infection, Confocal Microscopy, Quantitative RT-PCR, Transfection, Incubation, Transmission Assay, Electron Microscopy, Two Tailed Test