Journal: Molecular & Cellular Proteomics : MCP

Article Title: SARS-CoV-2 variants show different host cell proteome profiles with delayed immune response activation in Omicron-infected cells

doi: 10.1016/j.mcpro.2023.100537

Figure Lengend Snippet: Phosphorylation of IRF-3 and NF-kB upon infection with SARS-CoV-2 variants. Calu-3 cells were infected with SARS-CoV-2 variants at a MOI of 1 for 24 h. Representative immunoblot and quantification of (A) IRF-3 and pIRF-3 (S386), (B) Total NF-κB, NF-κB (p65) phosphorylation and Iκb alpha. Statistics were determined by two-way ANOVA (n = 3 biologically independent replicates), bars represent the mean and error bar shows ± SD/ s.e.m, * p<0.5, ** p<0.01, ns: not significant.

Article Snippet: Antibodies used were as followed: rabbit anti IRF-3 (Cell Signaling #4302 1:1000), rabbit anti IRF-3 (S386) (Cell Signaling #37829 1:1000), goat anti mouse HRP (Jackson Immunoresearch #115-035-062 1:10000), goat anti rabbit HRP (Merck #A6154 1:10000).

Techniques: Infection, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: SARS-CoV-2 variants show different host cell proteome profiles with delayed immune response activation in Omicron-infected cells

doi: 10.1016/j.mcpro.2023.100537

Figure Lengend Snippet: Kinetics of innate immune response induction upon infection with different SARS-CoV-2 variants . (A) Experimental scheme. Calu-3 cells were infected with different SARS-CoV-2 variants at a MOI of 0.01 for 24 h, 48 h and 72 h and immune response monitored. (B) Nuclear translocation of IRF-3 and NF-κB was quantified with immunofluorescence using the Harmony software (PerkinElmer). (C) Representative immunofluorescence images of IRF-3 and NF-κB stained cells at 48 hpi (blue: DAPI, green: IRF-3 or NF-κB, red: spike, scale bar represents 200 μm). (D) Interferon release was measured with HEK blue reporter cells for INFαß and INFλ release. Statistics of (B) and (D) were determined by two-way ANOVA (n = 3 biologically independent replicates), bars represent the mean and error bar shows ± SD. **** p < 0.0001.

Article Snippet: Antibodies used were as followed: rabbit anti IRF-3 (Cell Signaling #4302 1:1000), rabbit anti IRF-3 (S386) (Cell Signaling #37829 1:1000), goat anti mouse HRP (Jackson Immunoresearch #115-035-062 1:10000), goat anti rabbit HRP (Merck #A6154 1:10000).

Techniques: Infection, Translocation Assay, Immunofluorescence, Software, Staining

Journal: Frontiers in Immunology

Article Title: Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection

doi: 10.3389/fimmu.2023.930086

Figure Lengend Snippet: Human coronavirus infection activates IRF1, IRF3 and IRF7. MRC5 cells were left uninfected (-) or infected (+) with 229E (A) or OC43 (B) at MOI of 0.01. (C) Vero E6 cells expressing TMPRSS2 were left uninfected or infected with SARS-CoV-2 at MOI of 0.01 or at MOI 0.1. The activation of IRF1, IRF3 and IRF7 was determined by western blot analysis using antibodies against 229E N protein, OC43 N protein, SARS-CoV-2 N protein, IRF1, IRF3, IRF7, phosphorylated (p-IRF3) and GAPDH.

Article Snippet: Antibodies used in this study include: IRF3, phospho-IRF3, IRF7, phsopho-STAT1 (Cell signalling technology), IRF3 (Santa Cruz), IRF1 (BD Transduction Laboratories), GAPDH (Santa Cruz Biotechnology), 229E N protein (Ingenasa), OC43 N protein (Millipore), SARS-CoV-2 spike protein (Sino Biological).

Techniques: Infection, Expressing, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection

doi: 10.3389/fimmu.2023.930086

Figure Lengend Snippet: Knockdown of IRFs promotes human coronavirus infection. MRC5 cells were transfected with control siRNA (siCont), IRF1 siRNA, IRF3 siRNA or IRF7 siRNA oligoes (5 pmol) using Lipofectamine RNAiMAX transfection reagent. The knockdown of IRFs expression was confirmed by western blot analysis for IRF1 and IRF3 (A) and RT-qPCR for IRF7 (B) . MRC5 cells were then infected with 229E or OC43 at MOI of 0.01. (C) The amounts of viral RNA were measured by RT-qPCR (n=3). (D) The amounts of viral protein were determined by western blot analysis using antibodies against 229E N protein, OC43 N protein and GAPDH. For RT-qPCR analysis, the transcription level for each gene was first normalized to GAPDH expression level. The fold change to control indicates the fold change of the expression level for IRFs siRNA transfected samples to that of the control siRNA transfected samples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Two-way ANOVA.

Article Snippet: Antibodies used in this study include: IRF3, phospho-IRF3, IRF7, phsopho-STAT1 (Cell signalling technology), IRF3 (Santa Cruz), IRF1 (BD Transduction Laboratories), GAPDH (Santa Cruz Biotechnology), 229E N protein (Ingenasa), OC43 N protein (Millipore), SARS-CoV-2 spike protein (Sino Biological).

Techniques: Infection, Transfection, Expressing, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection

doi: 10.3389/fimmu.2023.930086

Figure Lengend Snippet: Overexpression of IRFs inhibits human coronavirus infection. H1299 cells were transfected with control pcDNA3 empty plasmid (EV) or the plasmid containing IRF1 (A) , IRF3 (B) or IRF7 (C) and then infected with or without 229E or OC43 at MOI of 0.01. Virus infection was determined by western blot analysis using antibodies against 229E N protein, OC43 N protein, IRF1, IRF3, IRF7, phosphorylated IRF3 (p-IRF3) and GAPDH. Amounts of progeny viruses were measured in the supernatant of cells transfected with the plasmid containing IRF1 (D) , IRF3 (E) or IRF7 (F) by TCID 50 assay which were compared to those transfected with empty plasmid (n=6). *p<0.05, **p<0.01, ***p<0.001, Two-way ANOVA.

Article Snippet: Antibodies used in this study include: IRF3, phospho-IRF3, IRF7, phsopho-STAT1 (Cell signalling technology), IRF3 (Santa Cruz), IRF1 (BD Transduction Laboratories), GAPDH (Santa Cruz Biotechnology), 229E N protein (Ingenasa), OC43 N protein (Millipore), SARS-CoV-2 spike protein (Sino Biological).

Techniques: Over Expression, Infection, Transfection, Plasmid Preparation, Western Blot

Journal: Frontiers in Immunology

Article Title: Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection

doi: 10.3389/fimmu.2023.930086

Figure Lengend Snippet: IRF3 effectively activates transcription of antiviral genes during human coronavirus infection. H1299 cells were transfected with control pcDNA3 plasmid (Cont) or the plasmid containing IRF1, IRF3 or IRF7 and then infected with or without 229E or OC43 at MOI of 0.01. (A) The expression of IRF1, IRF3 and IRF7 was confirmed by western blot analysis. At 24 hours after infection, the expression of GBP2, IFI2, MAP2, RIG-I, STAT2 and IFN-β in the cells infected with 229E (B) or OC43 (C) was determined by RT-qPCR (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Two-way ANOVA.

Article Snippet: Antibodies used in this study include: IRF3, phospho-IRF3, IRF7, phsopho-STAT1 (Cell signalling technology), IRF3 (Santa Cruz), IRF1 (BD Transduction Laboratories), GAPDH (Santa Cruz Biotechnology), 229E N protein (Ingenasa), OC43 N protein (Millipore), SARS-CoV-2 spike protein (Sino Biological).

Techniques: Infection, Transfection, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR

Journal: Signal Transduction and Targeted Therapy

Article Title: Phase-separated nucleocapsid protein of SARS-CoV-2 suppresses cGAS-DNA recognition by disrupting cGAS-G3BP1 complex

doi: 10.1038/s41392-023-01420-9

Figure Lengend Snippet: SARS-CoV-2 nucleocapsid protein counteracts cGAS-mediated IFN-I signaling by dampening cGAS-DNA recognition. a HEK-293T cells transfected with IFNβ luciferase reporter (IFNβ-luc) and TK-luc, Flag-GFP-cGAS and HA-STING, along with plasmids expressing SARS-CoV-2 viral protein were treated with HT-DNA for 12 h. Cell lysates were collected and luciferase assay was performed. b HEK-293T cells were transfected with IFNβ-luc, TK-luc, Flag-GFP-cGAS and HA-STING, along with increasing expressed SARS-CoV-2 N protein. Cell lysates were collected and luciferase assay was performed. c HEK-293T cells were transfected with IFNβ-luc, TK-luc, plasmids expressing cGAS, STING, TBK1 or IRF3 (5D), along with Flag-empty vector (EV) or Flag-N. Cell lysates were collected and luciferase assay was performed. d Immunoblot analysis of N protein-inducible A549 cells treated with doxycycline (Dox, 200 ng/mL) for 24 h or left untreated (UT) with ISD (2 μg/mL) treatment for indicated time points. e Immunoblot analysis of N protein-inducible A549 cells treated with doxycycline (Dox, 200 ng/mL) for 24 h or left untreated with HT-DNA (2 μg/mL) treatment for indicated time points. f N protein-inducible Calu3 cells treated with increasing concentration of Dox (200 ng/mL and 400 ng/mL) were transfected with mCherry plasmid (2 μg/mL) as plasmid DNA for 2 h before harvest. Cell lysates were collected, immunoprecipitated with A + G beads together with cGAS antibody, followed by qRT-PCR analysis of extracted DNA to detect cGAS-bound plasmid DNA ( mCherry ) abundance. g N protein-inducible Calu3 cells treated with increasing concentration of Dox (200 ng/mL and 400 ng/mL) and transfected with biotin-ISD (2 μg/mL) stimulation for 2 h before harvest. Cell lysates were collected, immunoprecipitated with NeutrAvidin agarose resin, followed by immunoblotting analysis. h N protein-inducible Calu3 cells treated with Dox (200 ng/mL) or left UT were infected by SARS-CoV-2 (MOI = 0.1) for indicated time points. Cell lysates were collected, immunoprecipitated with A + G beads together with cGAS antibody, followed by qRT-PCR analysis of extracted DNA to detect cGAS-bound mtDNA abundance. Data in a – c , f , h ) were expressed as mean ± SEM of 3 independent biological experiments. **** P < 0.0001, ns not significant (unpaired two-tailed student’s t -test). Similar results were obtained for 3 independent biological experiments in d , e , g

Article Snippet: Anti-phospho-STAT1 (Tyr701), anti-STAT1, anti-phospho-TBK1 (Ser172), anti-TBK1, anti-phospho-IRF3 (Ser396) and anti-IRF3 were purchased from Cell Signaling Technology.

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Western Blot, Concentration Assay, Immunoprecipitation, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 deficiency inhibits VSV-induced IKKε and IRF3 phosphorylation. a , b Immunoblot analysis of phosphorylated and total proteins in lysates of PMs obtained from WT (TAOK1 f/f ) and TAOK1cko (Lyz2 + TAOK1 f/f ) mice infected with VSV for indicated time. c Immunoblot analysis of phosphorylated and total proteins in lysates of Myc-TAOK1 overexpressing RAW264.7 macrophages infected with VSV for indicated time. d Luciferase activity in HEK293T cells transfected with Ifnb luciferase reporter, a Renilla-TK reporter, and an expression vector for TAOK1, along with plasmids encoding RIG-I N, TBK1, IKKε, and IRF3 5D. Data are representative of three independent experiments. Data are mean ± SD. * p < 0.05, ** p < 0.01 (Student's t test). ns, not significant.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Infection, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 interacts with TBK1. a Mouse PMs (from wild-type [WT] C57BL/6 mice, 6–8 weeks old) were infected with VSV for indicated hours. Immunoblot analysis of endogenous signal adapters immunoprecipitated with antibody to TAOK1. b Immunoblot analysis of HEK293T cells that co-transfected with Myc-TAOK1 plus HA-TBK1 or HA-IRF3 plasmids, then immunoprecipitated with antibody to HA tag. c Immunoblot analysis of HEK293T cells that co-transfected with Myc-TAOK1 plus Flag-RIG-I plasmids, then immunoprecipitated with the antibody to Flag tag. d Immunoblot analysis of HEK293T cells that co-transfected with Myc-TAOK1 plus HA-MAVS plasmids, then immunoprecipitated with the antibody to HA tag. e WT and MAVS-deficient HEK293T (MAVS-KO) cells were infected with VSV for indicated time, followed by immunoprecipitation (IP) with anti-TAOK1 and immunoblot analysis with indicated antibodies. f Q-PCR analysis of IFNB1 and TNFA mRNA expression in MAVS-deficient HEK293T (MAVS-KO) cells transfected with TAOK1 expressing plasmid and infected with VSV for indicated time. Data are from one experiment of three similar results. Data are mean ± SD. * p < 0.05, ** p < 0.01 (Student's t test). WCL, whole-cell lysates; nd, not detected.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Western Blot, Immunoprecipitation, Transfection, FLAG-tag, Expressing, Plasmid Preparation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 promotes TBK1-IRF3 complex formation. a HEK293T cells were transfected with plasmids encoding HA-TBK1, Flag-IRF3, and varying doses of a plasmid encoding Myc-TAOK1 (0.5, 1.0, and 1.5 μg). Cells were harvested 24 h after transfection for immunoblot analysis as indicated with the antibody to Flag tag. b HEK293T cells were transfected with plasmids encoding Flag-IKKε, HA-IRF3, and varying doses of a plasmid encoding Myc-TAOK1 (0.5, 1.0, and 1.5 μg). Cells were harvested 24 h after transfection for immunoblot analysis as indicated with antibody to HA tag. c Mouse PMs from WT (TAOK1 f/f ) and TAOK1cko (Lyz2 + TAOK1 f/f ) mice were infected with VSV for indicated hours. Immunoblot analysis of endogenous IRF3 immunoprecipitated with antibody to TBK1. Data are from one experiment of three similar results. * p < 0.05, ** p < 0.01. WCL, whole-cell lysates; ns, not significant.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, FLAG-tag, Infection, Immunoprecipitation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 promotes the interaction between TBK1 and IRF3 by trafficking TBK1 along microtubules. a HeLa cells co-transfected with Flag-IRF3 and Myc-TAOK1 plasmids, then pretreated with DMSO or colchicine (10 μm) for 1 h, followed by infection with or without VSV for 4 h. Confocal microscopy of co-localization between Flag-IRF3 (green) and endogeneous TBK1 (red). DAPI served as a marker of nuclei (blue). Scale bar, 10 μm. b Confocal microscopy of HeLa cells transfected with Myc-TAOK1, Myc-TAOK1 K57A, Myc-TAOK1C, and Myc-TAOK1N plasmids (green) followed by VSV infection for 4 h α-tubulin (red) was used to probe the microtubules. DAPI served as a marker of nuclei (blue). Scale bar, 10 μm. c Immunoblot analysis of phosphorylated and total MAP4 proteins in lysates of PMs obtained from WT (TAOK1 f/f ) and TAOK1cko (Lyz2 + TAOK1 f/f ) mice infected with VSV for indicated time. d Mouse PMs were pretreated with DMSO or colchicine (10 μm) for 1 h and then infected with VSV for 4 h. Immunoblot analysis of phosphorylated and total IRF3 and MAP4 proteins in cell lysates. e Mouse PMs were pretreated with DMSO or colchicine (10 μM) for 1 h and then infected with VSV for 4 h. Immunoblot analysis of endogenous signal adapters immunoprecipitated with antibody to TAOK1. * p < 0.05, ** p < 0.01. Data are representative of three independent experiments with similar results. ns, not significant; WCL, whole-cell lysates.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Infection, Confocal Microscopy, Marker, Western Blot, Immunoprecipitation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: A schematic diagram revealing the proposed mechanism by which TAOK1 positively regulates antiviral immune responses. (1) TAOK1 promoted virus-induced MAP4 phosphorylation and microtubule detachment; (2) TAOK1 bound more dynein instead of MAP4 upon virus infection and promoted the perinuclear transport of TBK1 along microtubules; (3) TAOK1 positively regulated antiviral signaling by promoting the TBK1-IRF3 interaction and IRF3 phosphorylation.

Article Snippet: Antibodies specific for p-IKKε (Ser172) (#8766), IKKε (#3416), p-IRF3 (Ser396) (#4947), p-NF-κB p65 (#3033), NF-κB p65 (#8242), p-TBK1 (Ser172) (#5483), TBK1 (#3504), p-ERK (#4370), ERK (#4695), p-JNK (#4668), JNK (#9285), p-p38 (#9215), p38 (#8690), and MAVS (#4983) and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection

Journal: Nature nanotechnology

Article Title: Immunological conversion of solid tumours using a bispecific nanobioconjugate for cancer immunotherapy

doi: 10.1038/s41565-022-01245-7

Figure Lengend Snippet: a, Tumor growth was inhibited by the triple-combination treatment and significantly prolonged the survival of EO771/E2 tumor-bearing mice (n=7). b, STING knockout partially abrogated the antitumor effect of the triple-combination treatment in EO771/E2-bearing mice (n=5). c-d, Immunofluorescence staining of EO771/E2 tumors implanted in wild-type (WT) (c) and STING knockout (d) mice (scale bar, 50 μm). Areas within the dashed squares are shown at higher magnification in the images to the right; arrows indicate the nuclear-translocated pIRF3 within F4/80+ macrophage or CD11c+ dendritic cells (scale bar, 20 μm). e, Quantification of infiltrated F4/80+ macrophages and percentage of nuclear pIRF3+ macrophages in the tumors (n=5). f, Expression of type I interferons is elevated in intratumoral F4/80+ macrophages after triple combination treatment (IFNA, interferon α; IFNB, interferon β) (n=4 for WT, n=3 for STING KO). For all figures, data are presented as mean±s.e.m.; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-tailed unpaired Student’s t-test for the indicated comparisons in figure 4a,​,b,b, ​,ee and ​andf;f; *P<0.05, ****P<0.0001 by log-rank test; n.s., not significant.

Article Snippet: For the immunostaining studies, tumour slides were pre-blocked with a blocking solution containing 5% BSA and 10% control normal serum and then stained with primary antibodies including CD4 (clone 4SM95, eBioscience, #14-9766-82, 1:50), CD8 (clone 4SM15, eBioscience, #100702, 1:50), F4/80 (clone CI:A3-1, Abcam, #6640, 1:100), Iba-1 (pAb, Abcam, #ab5076, 1:1000), CD11c (clone N418, eBioscience, #14-0114-82, 1:100), pIRF3 S396 (clone D6O1M, Cell Signaling, #29047, 1:50), PD1 (clone D7D5W, Cell Signaling, #84651, 1:200), and PDL1 (clone D5V3B, Cell Signaling, #64988, 1:200) at 4 °C overnight.

Techniques: Knock-Out, Immunofluorescence, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: The M35 protein forms homodimers after crystallisation and in cell lysates. (A) Ribbon representations of the M35 protein crystal structure. M35_S (aa 2-452) was crystallised and its structure solved at 1.94 Å. M35 monomers are depicted in red (aa 7-343 and aa 377-441) or orange (aa 8-345 and aa 376-440), respectively. Visible N and C termini (bold) and the ends of each protein chain are labelled accordingly. The structure is depicted from three perspectives. (B) Size-exclusion chromatography followed by multi-angle light scattering (SEC- MALS) of purified NStr-M35_S protein. LS: light scattering. (C) Co-immunoprecipitation of M35 in cell lysates. HEK293T were co-transfected with indicated expression plasmids for M35-V5/His and M35-HAHA, M34-V5/His and M35-HAHA (negative control), or single constructs filled up with EV. An anti-V5-immunoprecipitation (IP) was performed 24 h later. Input and IP samples were analysed by SDS-PAGE and immunoblotting with HA- and V5-specific antibodies. Detection of GAPDH served as loading control, # unspecific band. One representative of two independent experiments is shown. (D) Native PAGE of M35 in cell lysates. HEK293T cells were co-transfected with expression plasmids for eGFP-IRF3 (control) or M35-V5/His or the corresponding EV, and for Flag-MAVS (stimulated conditions) or the respective EV (unstimulated conditions). Cells were lysed 20 h later and analysed in parallel by native (upper panel) or SDS-PAGE (lower panel) followed by immunoblotting and detection with GFP-, V5-, Flag- and GAPDH-specific antibodies as indicated. Lysates of M35-V5/His-expressing cells were diluted 1:4 in lysis buffer to adjust the signal strength in the native immunoblot. One representative of three independent experiments is shown.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Size-exclusion Chromatography, Purification, Immunoprecipitation, Transfection, Expressing, Negative Control, Construct, SDS Page, Western Blot, Clear Native PAGE, Lysis

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: Identified M35 loss-of-function mutations impair the homodimerization of M35. (A) Immunofluorescence assay of M35 derivatives. HEK293T cells transfected with expression constructs for M35-V5/His WT, Δβ, or R69A or the corresponding EV were subjected to immunofluorescence labelling with a V5-specific antibody. Nuclei were stained with Hoechst. The scale bar represents 10 µm. Images are representative of at least two independent experiments. (B) Native PAGE of M35 derivatives. Native (upper panel) and SDS-PAGE (lower panel) followed by immunoblotting were performed as described before by co-transfecting HEK293T with expression plasmids for eGFP-IRF3, or M35-V5/His WT, Δβ, or R69A or the respective EV, and for Flag-MAVS (stimulated conditions) or the respective EV (unstimulated conditions), and analysis with GFP-, V5-, Flag- and GAPDH-specific antibodies. Lysates with M35-WT and M35-Δβ were diluted as indicated in lysis buffer to adjust the signal strength in the native immunoblot. One representative of three independent experiments is shown.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Immunofluorescence, Transfection, Expressing, Construct, Staining, Clear Native PAGE, SDS Page, Western Blot, Lysis

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: Presence of M35 impairs binding of IRF3 to the host’s IFNβ enhancer upon stimulation of PRR signalling. (A) Chromatin immunoprecipitation (ChIP) assay. iMEFs stably expressing M35-myc/His or the corresponding EV were stimulated by transfection of poly(I:C) or mock-treated. After 6 h, formaldehyde (FA) was applied to cross-link interactions and cells were harvested. Chromatin was isolated, fragmented for processing, and subjected to immunoprecipitation with an IRF3- specific antibody. The precipitated material was decrosslinked, DNA was purified and analysed by qPCR alongside 1% of input material. (B) Immunoblot of chromatin samples from iMEFs. iMEFs were processed as described in (A) and analysed by immunoblotting with myc-, pIRF3-, and IRF3- specific antibodies, and fibrillarin-specific antibodies. Fibrillarin served as a loading control for the nuclear fraction. Shown is one representative of three independent experiments. (C) ChIP for recruitment of IRF3 to the Ifnb1 promoter in presence or absence of M35. ChIP was performed as described in (A) with an IRF3-specific and an IgG control antibody, and samples were analysed for enrichment of the IFNβ enhancer sequence by qPCR. A primer set targeting the promoter of Il6 upstream of a predicted IRF3 binding site was used as negative control. Shown are combined data from two independent experiments.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Stable Transfection, Expressing, Transfection, Isolation, Immunoprecipitation, Purification, Western Blot, Sequencing, Negative Control

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: SLAM-seq for characterisation of the dependency of ISD-stimulated transcripts on IRF3 or of IFNβ-stimulated transcripts on canonical type I IFN-IFNAR1/IFNAR2 signalling in MEFs. (A) Determination of IRF3-dependent and IFNAR1-responsive transcripts. Primary MEFs of WT, IRF3 -/- or IFNAR1 -/- mice were stimulated by transfection of 5 µg/mL ISD for 4 h or mock- transfected, or stimulated with 100 U/mL of murine IFNβ for 3 h or left untreated. Transcripts were labelled in the last 2 h of stimulation by incubation with 200 µM of 4-thiouridine (4sU) and analysed by SLAM-seq. Samples were prepared and analysed in quadruplicate. (B-C) Heatmaps showing the log fold-changes (log FC; blue: down-, red: up-regulation) in the indicated cell lines for (B) the 28 genes IRF3-dependent genes detected after ISD stimulation or (C) the 2,888 IFNAR1-responsive genes detected after IFNβ treatment. Transcripts with an FDR ≤ 0.01 were considered statistically significant. The green marks on the left indicate overlaps with IFNα- responsive genes in human fibroblasts or conserved (core) between ten different species , brown marks show significant regulation in the different cells. Genes were clustered according to Euclidean distances with Ward’s clustering criterion. (D) Venn diagram showing overlaps of genes regulated in an IRF3-specific manner in response to ISD treatment (IRF3-dependent genes), regulated upon IFNβ in WT MEFs (independent of regulation in IFNAR1 -/- cells) or regulated by IFNβ only in WT but not IFNAR1 -/- MEFs (IFNAR1-responsive genes). (E) Correlation plot showing spearman correlation (blue: negative, red: positive correlation) for pairwise comparisons of log FC for indicated treatments and cell lines for IRF3-dependent genes. (F-G) Heatmaps showing the log FC (blue: down-, red: up-regulation) of (F) IRF3-dependent or (G) IFNAR1-responsive genes in WT cells after IFNβ or ISD treatment compared to controls, and in untreated knockout cell lines compared to WT. Genes significantly differentially expressed (FDR ≤ 0.01) in the IFNAR1 -/- or IRF3 -/- compared to WT MEFs are marked on the left (blue: down-, red: up- regulation).

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Transfection, Incubation, Knock-Out

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: Presence of M35 modulates expression of IRF3-dependent genes. (A) Determination of the global effect of M35’s presence on gene expression. iMEFs stably expressing M35-HAHA or a corresponding EV were stimulated by transfection of 5 µg/mL ISD, mock-transfected or left untreated and incubated for indicated times. Transcripts were labelled in the last 90 min of stimulation by incubation with 200 µM of 4-thiouridine (4sU). Total transcripts were analysed by SLAM-seq. Samples were prepared and analysed in triplicate. (B) Depicted are log FCs of transcripts of EV (x axis) vs. M35-HAHA (y axis) iMEFs after indicated times of ISD stimulation compared to mock-transfection. (C) Expression kinetics of selected transcripts upon PRR stimulation in EV and M35-HAHA iMEFs. Total RNA counts are given in transcripts per million (tpm). Differences between transcript levels in ISD-stimulated EV and M35-HAHA iMEFs with FDR < 0.01 were considered statistically significant, ns non-significant. (D) Volcano plot showing differential expression of total cellular transcripts in EV compared to M35- HAHA iMEFs in untreated conditions as log FC (x axis), plotted against -log 10 of the FDR (y axis, with significantly (FDR < 0.01) regulated transcripts above the dashed horizontal line). Numbers indicate total up- (log FC > 0) or down-regulated (log FC < 0) transcripts in the respective sections. (E) Heatmaps showing the log FC (blue: down-, red: up-regulation) in the indicated SLAM-seq samples for the 28 IRF3-dependent genes. Genes differentially expressed (FDR ≤ 0.01) in M35-expressing compared to EV iMEFs or in IFNAR1 -/- or IRF3 -/- compared to WT MEFs are marked at the left (blue: down-, red: up-regulation). (F) Response of IRF3-dependent genes upon infection with MCMV with or without M35. Immortalised BMDMs (iBMDMs) pre- treated with 1 µM ruxolitinib (IFNAR signalling inhibitor) were infected with MCMV M35stopRevertant (REV) or MCMV M35stop (M35stop) at MOI of 0.1 or mock infected. Cells were harvested 4 h post infection for RT-qPCR analysis. Relative fold induction of Ifnb1 , Ifna4 , Ifit3 , and Rsad2 transcripts was calculated based on the housekeeping gene Rpl8 , and values were normalised to REV-infected samples. Data is shown as mean ±SD and combined from two ( Ifna4 ) or three ( Ifnb1 , Ifit3 , Rsad2 ) independent experiments. Significance compared to infection with REV was calculated by Student’s t -test (unpaired, two-tailed), ns non-significant, * p < 0.05, ** p < 0.01.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Expressing, Stable Transfection, Transfection, Incubation, Infection, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

doi: 10.1101/2023.03.21.533612

Figure Lengend Snippet: M35 binds to specific host promoters and interferes with IRF3-dependent gene expression. Upon infection of a host cell with MCMV, pathogen-associated molecular patterns (PAMPs) are sensed by pattern recognition receptors (PRRs) and activate the transcription factors NF-κB and IRF3. NF-κB induces expression of proinflammatory cytokines, NF-κB and IRF3 together induce expression of Ifnb1 , and IRF3 regulates expression of further type I interferons and induces a subset of the interferon-stimulated genes (ISGs). Released type I interferons activate the type I interferon receptor (IFNAR). IFNAR signalling induces assembly of different transcription factors complexes, mainly interferon-stimulated gene factor 3 (ISGF3) which further drives expression of various ISGs. During MCMV infection, the viral tegument protein M35 is released and rapidly shuttles to the nucleus. M35 binds to IRF3-targeted recognition elements in host promoters and thus antagonizes recruitment of IRF3, resulting in inhibition of IRF3-driven gene expression.

Article Snippet: Murine anti-myc- tag (clone 9B11, #2276), rabbit anti-phospho-IRF3 (clone 4D4G, Serine 396, #4947), rabbit anti- fibrillarin (clone C13C3, #2639), anti-GAPDH (clone 14C10, #2118), rabbit anti-HA-tag (clone C29F4, #3724) antibodies for immunoblots were purchased from Cell Signaling Technology (Frankfurt am Main, Germany).

Techniques: Expressing, Infection, Inhibition