Journal: Cell

Article Title: Serotonin reduction in post-acute sequelae of viral infection

doi: 10.1016/j.cell.2023.09.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse IFNAR-1 antibody , Bio X Cell , BE0241; RRID:AB_2687723.

Techniques: Virus, Clinical Proteomics, Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bicinchoninic Acid Protein Assay, SYBR Green Assay, TaqMan Assay, Software, Imaging, Control, Electron Microscopy, Low Protein Binding, Membrane

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) Human KR-2 cells were infected with HSV (MOI 0.1) and harvested at 0, 24, 26, 28, and 30 h post-infection. Cell lysates were immunoprecipitated (IP) using the anti-IFNAR1 (“R1”) antibody. The phosphorylation of IFNAR1 at Ser535 and total levels of IFNAR1 were analyzed by immunoblotting (IB) using the indicated antibodies. (B) MEF (from wild type or S526A mice) were uninfected (red line) or infected (blue line) with HSV (MOI 0.1 for 32 h). Cells were subsequently incubated with the anti-mouse IFNAR1 antibody, biotin-conjugated goat-anti-mouse IgG and PE-streptavidine and then analyzed by FACS (BD Caliber). Gray area depicts a control reaction with an isotype antibody. (C) Human WT-5, KR-2 or U5A fibrosarcoma cells were either infected with HSV (MOI 0.1 for 22 h) or treated with human IFNβ (1000 IU/ml for 30 min). The analysis of IFNAR1 levels and phosphorylation in IFNAR1 immunoprecipitates was carried out using the indicated antibodies. The analyses of STAT1 phosphorylation and levels in whole cell lysates (WCL) are also shown. Exp., exposure. (D) Human WT-5, KR-2 or U5A fibrosarcoma cells were left intact or infected with HSV (MOI 0.1 for 30 h). Analyses of IFNAR1 levels in IFNAR1 immunoprecipitates and β-actin (as a loading control) in the supernatants of the immunoprecipitation reactions were carried out using indicated antibodies.

Article Snippet: Antibodies against p-STAT1, p-p38 (Cell Signaling), phospho-Ser532, phospho-Ser535 (or phospho-Ser523, phospho-Ser526, respectively, in the murine receptor) , , murine IFNAR1 (LeinCo), STAT1, p38, phospho-PERK (Santa Cruz), Flag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol) were used for immunoprecipitation (IP) and immunoblotting (IB) as described previously , .

Techniques: Infection, Immunoprecipitation, Phospho-proteomics, Western Blot, Incubation, Control

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) KR-2 cells were infected with HSV (MOI 0.1) and were treated with either DMSO (control) or CK1 inhibitor D4476 (12.5 µM for 1 h) before harvesting at 0, 21, or 23 h post-infection. Cell lysates were immunoprecipitated with the IFNAR1 antibody and analyzed by IB with the indicated antibodies. (B) KR-2 cells were infected with HSV (MOI 0.1) and were harvested at 22 or 24 h post-infection. Cell lysates were analyzed by IFNAR1 IP followed by IB using antibodies against ubiquitin, phospho-S532, and IFNAR1. (C) KR-2 cells expressing Flag-IFNAR1 (wild type or S532A mutant) were infected with HSV (MOI 0.1) and harvested 24 h post-infection. The phosphorylation and ubiquitination of Flag-IFNAR1 were analyzed by IP using anti-Flag antibodies followed by an IB analysis as described in panel D. (D) KR-2 cells expressing the indicated Flag-IFNAR1 (wild type or S532A mutant) were infected with HSV (MOI 0.1) and harvested 30 h post-infection. Levels of exogenous IFNAR1 and β-actin were analyzed by IB using the indicated antibodies.

Article Snippet: Antibodies against p-STAT1, p-p38 (Cell Signaling), phospho-Ser532, phospho-Ser535 (or phospho-Ser523, phospho-Ser526, respectively, in the murine receptor) , , murine IFNAR1 (LeinCo), STAT1, p38, phospho-PERK (Santa Cruz), Flag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol) were used for immunoprecipitation (IP) and immunoblotting (IB) as described previously , .

Techniques: Infection, Control, Immunoprecipitation, Ubiquitin Proteomics, Expressing, Mutagenesis, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) KR-2 cells were uninfected or infected with VSV (MOI 0.1 for 12 and 14 h) or HSV (MOI 0.1 for 14 and 30 h). Cell lysates were analyzed by IB using the indicated antibodies. (B) KR-2 cells were treated with thapsigargin (TG, 1 µM for 30 min) or infected with HSV (MOI 0.1 for 22 h). Phosphorylation and total levels of PERK were analyzed using the indicated antibodies. (C) KR-2 cells were transduced with lentiviral vectors that express shRNA against PERK or against GFP (shCON, used as a control) and treated or infected as described in panel B. The phosphorylation and levels of IFNAR1 in immunoprecipitates and phosphorylation and levels of PERK and β-actin in whole cell lysates (WCL) were analyzed using the indicated antibodies. (D) MEFs from PERK fl/fl mice were transduced with an empty lentivirus (Mock) or lentiviral vector for the expression of Cre recombinase. Cells were then left untreated (magenta line) or infected with VSV (MOI 0.1 for 14 h, blue line) or HSV (MOI 0.1 for 30 h, red line). Cell surface IFNAR1 levels were analyzed by FACS.

Article Snippet: Antibodies against p-STAT1, p-p38 (Cell Signaling), phospho-Ser532, phospho-Ser535 (or phospho-Ser523, phospho-Ser526, respectively, in the murine receptor) , , murine IFNAR1 (LeinCo), STAT1, p38, phospho-PERK (Santa Cruz), Flag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol) were used for immunoprecipitation (IP) and immunoblotting (IB) as described previously , .

Techniques: Infection, Phospho-proteomics, Transduction, shRNA, Control, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) The stock of HSV was inactivated or not with UV and used to treat KR-2 cells at MOI 5.0 for indicated time points. Cell lysates were analyzed by IP using the anti-IFNAR1 antibody and IB using indicated antibodies. (B) KR-2 cells were treated with either UV- or sham-treated HSV as shown in panel A. The levels of IFNAR1 and β-actin were assessed as in . (C) KR-2 cells were untreated or treated with UV-inactivated HSV (MOI 5.0) in the presence of cycloheximide (20 µg/mL) for indicated times. Analyses of total levels of IFNAR1 (assessed by IP-IB) and β-actin (as a loading control) are depicted. (D) KR-2 cells expressing the indicated Flag-IFNAR1 proteins were treated as shown in panel D. The levels of exogenous IFNAR1 were analyzed by IB using the anti-Flag antibody.

Article Snippet: Antibodies against p-STAT1, p-p38 (Cell Signaling), phospho-Ser532, phospho-Ser535 (or phospho-Ser523, phospho-Ser526, respectively, in the murine receptor) , , murine IFNAR1 (LeinCo), STAT1, p38, phospho-PERK (Santa Cruz), Flag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol) were used for immunoprecipitation (IP) and immunoblotting (IB) as described previously , .

Techniques: Control, Expressing

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) KR-2 cells were treated with CpG (10 µM for 30 min) or with HSV (5 MOI for 90 min). The phosphorylation and levels of IFNAR1 were detected after IFNAR1 immunoprecipitation using the indicated antibodies. The levels and activity of p38 kinase in WCL were also analyzed. (B) U937 cells were untreated or treated with UV-inactivated HSV (MOI 5), CpG (10 µM) or LPS (10 µg/mL) for 1 h. The phosphorylation and levels of IFNAR1 were analyzed by IP-IB using the indicated antibodies. (C) U937 cells were pre-treated with inhibitors of JNK (SP600125, 10 µM) or p38 kinase (SB203580, 10 µM) for 1 h and then treated with LPS (10 µg/ml for 1 h) and analyzed as shown in panel B. (D) U937 cells were untreated or pre-treated with the p38 kinase inhibitor SB203580 (10 µM for 1 h) and then treated with the indicated PRR agonists or UV-inactivated HSV (MOI 1), or UV-inactivated VSV (MOI 1) for 1 h and then analyzed as depicted in panel B.

Article Snippet: Antibodies against p-STAT1, p-p38 (Cell Signaling), phospho-Ser532, phospho-Ser535 (or phospho-Ser523, phospho-Ser526, respectively, in the murine receptor) , , murine IFNAR1 (LeinCo), STAT1, p38, phospho-PERK (Santa Cruz), Flag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol) were used for immunoprecipitation (IP) and immunoblotting (IB) as described previously , .

Techniques: Phospho-proteomics, Immunoprecipitation, Activity Assay

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) Lysates from KR-2 cells treated with UV-inactivated HSV (MOI 5 for 90 min) were used as a source of kinase activity in an in vitro assay measuring the phosphorylation of GST-IFNAR1 protein analyzed by IB using anti-pS532 antibody (upper panel) or anti-GST antibody (lower panel). The effects of the p38 kinase inhibitors (SB203580, 50 nM, or VX-702, 20 nM) or the JNK inhibitor (SP600125, 40 nM) are also shown. (B) In vitro phosphorylation of GST-IFNAR1 (wild type or S532A mutant) by recombinant active GST-p38α in the presence of γ- 32 P-ATP was determined by conducting a SDS-PAGE followed by an autoradiography. The levels of GST-IFNAR1 were analyzed by Coomassie Blue staining (CB). (C) KR-2 cells were transfected with either empty vector (pcDNA3) or Flag-p38α (WT or AGF mutant) as indicated. Cells were either left untreated or treated with UV-inactivated HSV (MOI 5 for 90 min). p38α kinase was immunoprecipitated from the lysates with anti-Flag, and the immunoprecipitates were subject to an in vitro kinase assay using GST-IFNAR1 (“GST-R1”, WT, or S532A mutant) as substrates and monitored using the pS532 antibody (top panel). The amounts of GST-IFNAR1 and p38 in the IP reactions are shown. (D) U937 cells that received control shRNA or shRNA against p38α kinase were treated with LPS (10 µg/mL for 1 h) and harvested. The phosphorylation and levels of IFNAR1 were analyzed by IP-IB. The levels of p38α kinase in the whole cell lysates are also shown. (E) U937 cells were untreated or pre-treated with the p38 kinase inhibitor SB203580 (10 µM for 1 h) and then with either LPS (10 µg/mL for 1 h) or human IFNα (1000 IU/mL for 30 min) as indicated. The phosphorylation of IFNAR1 (by IP-IB) and of p38 kinase and STAT1 (in whole cell lysates by IB) was analyzed. (F) Cycloheximide chase analysis of endogenous IFNAR1 in U937 cells treated as in panel E was analyzed using the indicated antibodies. Levels and activation of p38 kinase were also determined.

Article Snippet: Antibodies against p-STAT1, p-p38 (Cell Signaling), phospho-Ser532, phospho-Ser535 (or phospho-Ser523, phospho-Ser526, respectively, in the murine receptor) , , murine IFNAR1 (LeinCo), STAT1, p38, phospho-PERK (Santa Cruz), Flag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol) were used for immunoprecipitation (IP) and immunoblotting (IB) as described previously , .

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Mutagenesis, Recombinant, SDS Page, Autoradiography, Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Kinase Assay, Control, shRNA, Activation Assay

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) Mouse bone marrow-derived macrophages (BMM) were pre-treated with LPS (5 µg/mL for 2.5 h) in the presence of either IFNα/β neutralizing antibodies (1000 U/mL) or the p38 kinase inhibitor SB203580 (10 µM) or both, as indicated. Cells were then washed and incubated with murine IFNβ (250 IU/mL for 30 min). The phosphorylation and levels of STAT1 are analyzed in . The asterisk denotes an experiment where neutralizing antibodies were added together with IFNβ. (B) BMM from wild type mice or mice expressing the IFNAR1 S526A mutant were treated and analyzed as indicated according to the description in panel A. (C) Mouse bone marrow-derived dendritic cells (BMDC) from wild type or IFNAR1-null mice were untreated or pre-treated with VX-702 (1 µM for 1 h) and then activated with LPS (2 µg/mL for 48 h). The percent of CD11c-positive and PI-negative viable DCs is depicted as an average of three independent experiments (± S.D.). The asterisk denotes p<0.05 in the t -test. (D) BMDC from wild type mice were untreated or pre-treated with VX-702 (1 µM for 1 h) and then treated with a neutralizing anti-IFNAR1 antibody (MAR, 10 µg/mL) or normal mouse IgG (mIgG) and activated by LPS (2 µg/mL for 48 h). The percent of CD11c-positive and PI-negative viable DCs is depicted as an average of three independent experiments (± S.D.). The asterisk denotes p<0.05 in the t -test. (E) Depicted is a hypothetical model that reflects a dual mode of regulation of the IFNα/β responses by PRR signaling (gray blunt arrows represent the inhibitory effects). The ligand-inducible pathway (that involves activation of TYK2 and PKD2) limits the extent of IFNα/β signaling in a cell that has been already exposed to these cytokines. Conversely, the ligand/JAK-independent pathway may (that involves PERK-dependent or independent activation of p38 kinase, priming phosphorylation and ensuing phosphorylation of IFNAR1 degron by CK1α) render a naïve cell less sensitive to its future encounters with Type I IFNs. The activation of PRR both induces production of endogenous Type I IFN and downregulates IFNAR1 in the very same IFN-producing cells. This mechanism may support the viability of the IFN-producing cells and temper an overall activity of IFNα/β pathways.

Article Snippet: Antibodies against p-STAT1, p-p38 (Cell Signaling), phospho-Ser532, phospho-Ser535 (or phospho-Ser523, phospho-Ser526, respectively, in the murine receptor) , , murine IFNAR1 (LeinCo), STAT1, p38, phospho-PERK (Santa Cruz), Flag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol) were used for immunoprecipitation (IP) and immunoblotting (IB) as described previously , .

Techniques: Derivative Assay, Incubation, Phospho-proteomics, Expressing, Mutagenesis, Activation Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation

doi: 10.3390/ijms23105773

Figure Lengend Snippet: Characterisation of UL34 as an augmenting viral protein with leaky late expression kinetics. ( A ) Intracellular HCMV genome copies at 120 HPI, relative to genome copies at 12 HPI. MOI = 3, n = 3, bars = SD. ( B ) Western blot analysis of UL34 expression in MRC5 cells treated with 100 μg/mL phosphonoacetic acid (PAA) and subsequently infected with either AD169-GFP WT or AD169 HA-UL34 HCMV (5 DPI, MOI = 3). Membranes were probed with primary antibodies against HCMV viral proteins, HA, or β-actin loading control. ( C ) Immuno-fluorescence analysis of host GM130 and viral UL99 in WT MRC5 cells infected with WT or ΔUL34 AD169-GFP virus. 4 DPI, MOI = 0.1, scale bars = 20 μm. ( D ) Growth kinetics of ΔUL34 AD169-GFP virus, as measured by IE1 fluorescent focus assay in cell culture supernatants from WT and UL34-complementing MRC5 cells. MOI = 3, n = 3, bars = SD. ( E ) Spread of ΔUL34 AD169-GFP virus in WT and UL34-complementing MRC5 cells, as quantified by fixing, staining, and counting IE1 positive cells at indicated time points. MOI = 0.01, n = 3, bars = SD.

Article Snippet: Membranes blocking was performed in 5% ( w / v ) skim milk in 1X TBST (150 mM NaCl, 50 mM Tris pH 7.4, 0.1% ( v / v ) Tween 20) at 4 °C for 1 h, membranes were incubated with primary antibodies: anti-UL99 (Clone 10B4 [ ]), anti-UL83 (Clone 8F5 [ ]), anti-MCP (custom ordered rabbit polyclonal, Cusabio, Houston, TX, USA), anti-HCMV gB (ab6499, Abcam, Cambridge, UK), anti-IE1 (Clone 1B12 [ ]), anti-UL26 [ ], anti-HA (ab130275, Abcam, Cambridge, UK), mouse anti-lamin A/C (4777T, Cell Signaling Technology, Danvers, MA, USA), rabbit phospho-lamin A/C (ser-22) (13448T, Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (A2228, Sigma, Burlington, MA, USA).

Techniques: Expressing, Western Blot, Infection, Fluorescence, Cell Culture, Staining