Journal: Virologica Sinica

Article Title: PRRSV degrades MDA5 via dual autophagy receptors P62 and CCT2 to evade antiviral innate immunity

doi: 10.1016/j.virs.2024.01.005

Figure Lengend Snippet: PRRSV can induce MDA5 to undergo selective autophagy degradation. A – B The schematic of the promoter design of the IFIH1 ( MDA5 ) gene. C 3D4/21 CD163 ​cells were transfected with MDA5-luc or a range of truncated constructs of MDA5 promoter. Luciferase activity was detected at 24 ​h post-transfection. D 3D4/21 CD163 ​cells were transfected with MDA5-4-luc, then infected by PRRSV. Luciferase activity in uninfected (Mock) or infected groups was determined at 12 or 24 ​h post-infection. E 3D4/21 CD163 ​cells were uninfected or infected with PRRSV (MOI of 0.5) for 24 ​h. MDA5 protein expression level was detected in immunoblot analysis. F MDA5 protein level in 3D4/21 CD163 ​cells treated with cycloheximide (CHX, 100 ​ng/mL) for the indicated times after uninfected or infected with PRRSV was determined by Western blotting analysis. The MDA5 intensity was quantified (normalized to β-Actin). G – H 3D4/21 CD163 ​cells were infected with PRRSV (MOI ​= ​0.5) and then treated with 50 ​μmol/L MG132 or 300 ​nmol/L Baf A1 for 6 ​h. The MDA5 and PRRSV N levels ( G ) in cells were subjected to qPCR analysis. The MDA5 protein level ( H ) in cell lysates were subjected to Western blotting analysis normalized to β-Actin. Data are representative of three independent experiments (means ​± ​SD). ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001, ns, no significance, two-tailed unpaired Student's t -test.

Article Snippet: Protease inhibitor MG132 (HY-13259), autophagy inhibitors chloroquine (HY-17589A) and bafilomycin A1 (HY-100558), autophagy activator rapamycin (HY-10219), and anti-c-Myc magnetic beads (HY-K0206) were obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transfection, Construct, Luciferase, Activity Assay, Infection, Expressing, Western Blot, Two Tailed Test

Journal: Virologica Sinica

Article Title: PRRSV degrades MDA5 via dual autophagy receptors P62 and CCT2 to evade antiviral innate immunity

doi: 10.1016/j.virs.2024.01.005

Figure Lengend Snippet: MDA5 also interacts with the novel autophagy receptor CCT2. A The structural diagram of the interaction between MDA5 and CCT2 protein was simulated by Cluspro. B HEK 293T cells transfected with EGFP-MDA5 (1 ​μg) together with Flag-CCT2 or CCT7 (1 ​μg), and then the anti-Flag beads were utilized for the coimmunoprecipitation and immunoblot analysis of the cell lysates. C HEK 293T cells were transfected with HA-CCT2 (1 ​μg), and then the anti-HA beads were utilized for the coimmunoprecipitation and immunoblot analysis for the cell lysates. D Co-localization analysis of MDA5 with CCT2. Myc-CCT2 (1 ​μg) and EGFP-MDA5 (1 ​μg) were transfected into HeLa cells. The cells were incubated with mouse anti-Myc mAb (red), and nuclei were stained with Hoechst (blue). Meanwhile, confocal laser scanning microscope was used to observe and image. Scale bars: 10 ​μm. E Coimmunoprecipitation and immunoblot analysis of the interaction of Myc-CCT2 (1 ​μg) together with Flag-MDA5 (N), Flag-MDA5 (M) or Flag-MDA5 (C) (1 ​μg) in the lysates of HEK 293T cells. F Coimmunoprecipitation and immunoblot analysis of the relation of EGFP-MDA5 (1 ​μg) with Flag-CCT2 (FL), Flag-CCT2 (ΔD1), Flag-CCT2 (ΔD2) or Flag-CCT2 (ΔD3) (1 ​μg) in the lysates of cells. G Coimmunoprecipitation and immunoblot analysis of the interaction between EGFP-LC3 (1 ​μg) with Flag-CCT2 (1 ​μg) in the lysates of HEK 293T cells. H Immunoblot analysis of lysates from cells transfected with Flag-MDA5 (1 ​μg) and an increasing amount of Myc-CCT2 (0, 0.25, 0.5 or 1 ​μg). I HEK 293T cells were transfected with Flag-MDA5 (1 ​μg), Myc-CCT2 (1 ​μg) and then subjected to separate processing with rapamycin (100 ​nmol/L), MG132 (50 ​μmol/L), Baf A1 (300 ​nmol/L) or CQ (50 ​μmol/L). The cell lysates were performed for immunoblot analysis.

Article Snippet: Protease inhibitor MG132 (HY-13259), autophagy inhibitors chloroquine (HY-17589A) and bafilomycin A1 (HY-100558), autophagy activator rapamycin (HY-10219), and anti-c-Myc magnetic beads (HY-K0206) were obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transfection, Western Blot, Incubation, Staining, Laser-Scanning Microscopy

Journal: Virologica Sinica

Article Title: PRRSV degrades MDA5 via dual autophagy receptors P62 and CCT2 to evade antiviral innate immunity

doi: 10.1016/j.virs.2024.01.005

Figure Lengend Snippet: Nsp3 enhances CCT2 interaction with MDA5 to promote degradation. A 3D4/21 CD163 ​cells were infected with PRRSV for 24 ​h, and CCT2 expression levels were detected in qPCR or immunoblot analysis. B Coimmunoprecipitation and immunoblot analysis of extracts of 3D4/21 CD163 ​cells transfected with EGFP-MDA5 (1 ​μg) and Flag-CCT2 (1 ​μg) and infected with or without PRRSV. C 3D4/21 CD163 ​cells were transfected with plasmids which empty vectors, PRRSV infectious clones or different nsps. The protein level of MDA5 was detected by Western blotting, 24 ​h after transfection. D Coimmunoprecipitation and immunoblot analysis of the interaction between Myc-nsp1β, nsp3 or N (1 ​μg) with Flag-MDA5 (1 ​μg) in the lysates of HEK 293T cells, using anti-Flag beads. E HEK 293T cells were transfected with Myc-nsp3 (1 ​μg), and then the anti-Myc beads were utilized for the coimmunoprecipitation and immunoblot analysis for the cell lysates. F Co-localization analysis of MDA5 with nsp3. Myc-nsp3 (1 ​μg) and EGFP-MDA5 (1 ​μg) were transfected into HeLa cells. The cells were incubated with mouse anti-Myc mAb (red), and nuclei were stained with Hoechst (blue), meanwhile, confocal laser scanning microscope was used to observe and image. Scale bars: 10 ​μm. G–H 3D4/21 CD163 ​cells were transfected with Myc-CCT2 (1 ​μg) and with or without Flag-MDA5 (1 ​μg), and then subjected to separate processing with rapamycin (100 ​nmol/L), MG132 (50 ​μmol/L), Baf A1 (300 ​nmol/L) or CQ (50 ​μmol/L), the cell lysates were performed to immunoblot analysis. I Coimmunoprecipitation and immunoblot analysis of lysates of HEK 293T cells transfected with EGFP-MDA5 (1 ​μg) and Flag-CCT2 (1 ​μg) and added with or without Myc-nsp1β, nsp3, nsp4 or nsp11 (1 ​μg). J Flag-MDA5 (1 ​μg), HA-CCT2 (1 ​μg) and an increasing amount of Myc-nsp3 (0, 0.5 or 1 ​μg) were cotransfected into cells for 24 ​h. Cell lysates were subjected to coimmunoprecipitation and immunoblot analysis with the indicated antibodies. K EGFP-MDA5 (1 ​μg), Flag-CCT2 (1 ​μg) and an increasing amount of Myc-nsp3 (0, 0.5, 1 or 2 ​μg) were cotransfected into HEK 293T cells for 24 ​h, and treated with Baf A1. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Data are representative of three independent experiments (means ​± ​SD). ∗∗, P ​< ​0.01, two-tailed unpaired Student's t -test.

Article Snippet: Protease inhibitor MG132 (HY-13259), autophagy inhibitors chloroquine (HY-17589A) and bafilomycin A1 (HY-100558), autophagy activator rapamycin (HY-10219), and anti-c-Myc magnetic beads (HY-K0206) were obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Infection, Expressing, Western Blot, Transfection, Clone Assay, Incubation, Staining, Laser-Scanning Microscopy, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

doi: 10.4103/1673-5374.379050

Figure Lengend Snippet: Fasudil-modified macrophages affect T cell polarization. A mouse model of chronic EAE was induced by subcutaneous injection of myelin oligodendrocyte glycoprotein peptde 35–55. Macrophages from EAE mice isolated with magnetic beads were treated with 15 μg/mL of Fasudil at 37°C in 5% CO 2 atmosphere for 72 hours. Fasudil-treated macrophages were suspended in PBS at a concentration of 2 × 10 7 cells/200 μL and intraperitoneally injected into EAE mice on days 3 and 11 postimmunization. PBS-treated macrophages were used as a control. The mice were randomly divided into two groups: the Fasudil-macrophage group (Fasudil-Mφ) ( n =12) and the PBS-macrophage group (PBS-Mφ) ( n = 12). On day 28 p.i., splenic MNCs were prepared and stained with anti-CD4 (for CD4 + T cells), IFN-γ (for Th1), IL-17 (for Th17), and IL-10 (for Th2) antibodies, and T cell subsets were analyzed by flow cytometry. Injection of Fasudil-modified macrophages decreased the percentage of CD4 + IL-17 + T cells and increased the percentage of CD4 + IL-10 + T cells. Quantitative results are shown for five mice from each group. The results are expressed as the percentage of double positive cells in the four-quadrant diagram. Data are presented as the mean ± SEM. ** P < 0.01, *** P < 0.001 (Student’s t -test). EAE: Experimental autoimmune encephalomyelits; IFN-γ: interferon-γ; IL-10:interleukin-10; IL-17:interleukin-17; MNCs: mononuclear cells; Mφ: macrophage; PBS: phosphate buffer saline.

Article Snippet: For intracellular staining, the MNCs were stained for 30 minutes at RT with the following antibodies diluted in 0.3% saponin/1% BSA-PBS buffer: the CD4 + T cell marker FITC-CD4 (Thermo Fisher Scientific, Cat# 11-0041-82, RRID: AB_464892), the Th1 marker PE-interferon (IFN)-γ (Thermo Fisher Scientific, Cat# 12-7311-82, RRID: AB_466193), the Th17 marker PE-IL-17 (Thermo Fisher Scientific, Cat# 12-7177-81, RRID: AB_763582), the Th2 marker PE-IL-10 (Thermo Fisher Scientific, Cat# 12-7101-82, RRID: AB_466176), the macrophage marker Alexa Fluor 488-anti-F4/80, the pro-inflammatory M1 macrophage subtype markers PE-IL-12 (Thermo Fisher Scientific, Cat# 12-7123-82, RRID: AB_466185), anti-iNOS, and anti-Arg-1, and the anti-inflammatory M2 macrophage subtype marker PE-IL-10, followed by staining with the corresponding secondary antibodies for iNOS and Arg-1.

Techniques: Modification, Injection, Isolation, Magnetic Beads, Concentration Assay, Control, Staining, Flow Cytometry, Saline