primary normal human bronchial epithelial (nhbe) cells  (Lonza)

Journal: mBio

Article Title: Targeting CTP synthetase 1 to restore interferon induction and impede nucleotide synthesis in SARS-CoV-2 infection

doi: 10.1128/mbio.00649-25

Figure Lengend Snippet: SARS-CoV-2 inhibits IFN induction via inducing IRF3 deamidation. ( A ) Normal human bronchial epithelial (NHBE) cells were infected with Sendai virus (SeV) (100 HAU/mL) or SARS-CoV-2 (MOI = 1). Total RNA was extracted, reverse-transcribed, and analyzed by real-time PCR with primers specific for IFNB1 , ISG15 , ISG56 , CCL5, and Mx1 . ( B ) NHBE cells were transfected with poly(I:C) and RNA purified from the medium of Vero E6-hACE2 cells infected with or without SARS-CoV-2 (72 hpi). The mRNA abundance of immune genes was analyzed by real-time PCR at 6 h post-transfection. ( C ) Modulation of IFN-β induction was determined by promoter activity in 293T cells expressing indicated SARS-CoV-2 proteins, with SeV infection. ( D ) Inhibition of antiviral gene expression by SARS-CoV-2 proteins in 293T cells infected with SeV was examined by real-time PCR with primers specific for indicated genes. ( E ) Inhibition of IFN-β induction by selected SARS-CoV-2 proteins was determined by reporter assay of 293T cells expressing TBK1 and IRF3. ( F ) Caco-2 cells were infected with SARS-CoV-2 for 48 h and 72 h with MOI 0.5. IRF3 charge status was analyzed by two-dimensional gel electrophoresis and immunoblotting. ( G ) Effects of selected SARS-CoV-2 proteins on IRF3 charge status and phosphorylation were determined by two-dimensional gel electrophoresis and immunoblotting analyses using lysates of 293T cells transfected with plasmids containing indicated genes. Data are presented as means ± SD of biological triplicates ( A–E ) and are representative of three independent experiments ( F and G ). Statistical significance was calculated using a one-way ANOVA test or unpaired, two-tailed Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Primary normal, human bronchial/tracheal epithelial (NHBE) cells were cultured in an airway epithelial cell medium according to ATCC’s recommendation.

Techniques: Infection, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Purification, Activity Assay, Expressing, Inhibition, Gene Expression, Reporter Assay, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Phospho-proteomics, Two Tailed Test

Journal: mBio

Article Title: Targeting CTP synthetase 1 to restore interferon induction and impede nucleotide synthesis in SARS-CoV-2 infection

doi: 10.1128/mbio.00649-25

Figure Lengend Snippet: CTPS1 negatively regulates SARS-CoV-2-mediated IFN induction. ( A ) CTPS1 depletion in NHBE cells was determined by immunoblotting. ( B through D ) Effects of CTPS1 depletion on the expression of cellular antiviral genes ( B ) and viral genes ( C ) were determined by real-time PCR analysis of total RNA extracted at 48 h after SARS-CoV-2 infection (MOI = 0.1). Medium of NHBE cells infected with SARS-CoV-2 was used for plaque assay to determine infectious viral progeny ( D ). ( E ) Knockout of CTPS1 in Caco-2 cells was analyzed by immunoblotting using cells from individual clones. ( F through H ) CTPS1 −/− Caco-2 cells were infected with SARS-CoV-2 for 24 h with MOI 0.5 ( F ) or 72 h with MOI 0.1 ( G and H ). Antiviral gene expression and viral RNA abundance in the cells were determined by real-time PCR ( F and G ). Viral titer in the medium was analyzed by plaque assay in Vero E6-hACE2 cells ( H ). ( I ) Interactions between endogenous CTPS1 and SARS-CoV-2 proteins were analyzed by co-immunoprecipitation in transfected 293T cells. Strep is a tag for SARS-CoV-2 proteins. ( J ) The effect of SARS-CoV-2 proteins on the charge status of IRF3-WT and IRF3-N85A was determined by two-dimensional gel electrophoresis and immunoblotting in wild-type and IRF3-N85A knock-in 293T cells. ( K through M ) Human ACE2-expressing Irf3 −/− Irf7 −/− MEFs were reconstituted with IRF3-WT, IRF3-N85D, IRF3-N85A, and vector. The effect of IRF3 and its mutants on Ifnb expression ( K ) and SARS-CoV-2 RNA abundance ( L ) was assessed by real-time PCR with total RNA extracted at 24 h after SARS-CoV-2 infection (MOI = 0.01). Medium of SARS-CoV-2-infected MEFs was used for plaque assay to determine infectious viral progeny ( M ). Data are presented as means ± SD of biological triplicates ( B through D, F through H, K through M ) and are representative of three independent experiments (A, E, I, and J). Statistical significance was calculated using the two-way ANOVA test, one-way ANOVA test, or unpaired, two-tailed Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Primary normal, human bronchial/tracheal epithelial (NHBE) cells were cultured in an airway epithelial cell medium according to ATCC’s recommendation.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Infection, Plaque Assay, Knock-Out, Clone Assay, Gene Expression, Immunoprecipitation, Transfection, Two-Dimensional Gel Electrophoresis, Electrophoresis, Knock-In, Plasmid Preparation, Two Tailed Test

Journal: mBio

Article Title: Targeting CTP synthetase 1 to restore interferon induction and impede nucleotide synthesis in SARS-CoV-2 infection

doi: 10.1128/mbio.00649-25

Figure Lengend Snippet: CTPS1 inhibitors impede SARS-CoV-2 replication. ( A ) Structures of compounds 1 and 2. ( B ) Effect of compound 1 on SARS-CoV-2 ORF8-induced IRF3 deamidation was analyzed by two-dimensional gel electrophoresis and immunoblotting in ORF8-expressing Caco-2 cells with compound 1 (5 μM) treatment for 4 h. ( C ) The effect of compound 1 on IFN induction by Sendai virus (SeV) infection was determined by luciferase reporter assay using control (CTL) or CTPS1-depleted 293T cells treated with increasing concentrations of compound 1. ( D ) FLAG-CTPS1 expressed 293T cells were treated with compound 2 at the indicated concentrations for 2 h. CTPS1 was purified and subjected to binding analysis by in-gel fluorescence imaging and Coomassie blue staining. ( E and F ) Effect of compound 1 on intracellular CTP or CDP synthesis was determined by [amide- 15 N]glutamine tracing and mass spectrometry using SARS-CoV-2 ORF8-expressing Caco-2 cells ( E ) or SARS-CoV-2-infected Caco-2 cells ( F ) treated with increasing concentrations of compound 1. M + a indicates targeted metabolites labeled with [amide- 15 N]. Metabolites with M + 2 are below the detection limit. ND, not detected. ( G ) Effect of compound 1 on CTPS1 activity in CTP synthesis, in the presence of 2 mM ATP, 2 mM L-glutamine, 0.1 mM GTP, and increasing concentrations of UTP, was determined by in vitro enzymatic assay and analyzed by mass spectrometry. Inhibition of CTP synthesis was normalized to DMSO control. ( H through J ) Caco-2 cells were treated with compound 1 and infected with SARS-CoV-2 (MOI = 0.1). The mRNA abundance of antiviral genes was determined by real-time PCR at 48 h after SARS-CoV-2 infection ( H ). The effect of compound 1 on SARS-CoV-2 RNA abundance ( I ) and infectious viral progeny ( J ) was determined at 72 h after SARS-CoV-2 infection by real-time PCR analysis of total RNA and plaque assay of the medium, respectively. ( K ) The effect of compound 1 and its derivatives on SARS-CoV-2 replication was determined by plaque assay at 72 h post-infection (MOI = 0.1) in the medium of Caco-2 cells. ( L and M ) Caco-2 ( L ) and NHBE cells ( M ) were treated with the indicated compounds and infected with SARS-CoV-2 (MOI = 0.1). Viral titer in the medium was determined by plaque assay. Effects of these compounds on cell viability were determined by XTT assay and plotted. IC 50 , IC 90 , and CC 50 were calculated. Data are presented as means ± SD of biological triplicates ( C, E, F, H through M ) and are representative of three independent experiments (B, D, and G). Statistical significance was calculated using the two-way ANOVA test or one-way ANOVA test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Primary normal, human bronchial/tracheal epithelial (NHBE) cells were cultured in an airway epithelial cell medium according to ATCC’s recommendation.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Expressing, Virus, Infection, Luciferase, Reporter Assay, Control, Purification, Binding Assay, Fluorescence, Imaging, Staining, Mass Spectrometry, Labeling, Activity Assay, In Vitro, Enzymatic Assay, Inhibition, Real-time Polymerase Chain Reaction, Plaque Assay, XTT Assay