Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA-mediated mitochondrial fragmentation is independent on the fission machinery proteins DLP1 and MFF. (A) Immunofluorescence analyses of MEFs MAVS-MITO cells: (a) control cells, (b) DLP1 silenced cells, (c) MFF silenced cells: (a–c) anti-TIM23; (d–f) overexpression of vMIA-Myc: (d) anti-TIM23, (e) anti-Myc, (f) merge image of d and e; (g–i) overexpression of vMIA-Myc in DLP1 silenced cells: (g) anti-TIM23, (h) anti-Myc, (i) merge image of g and h; (j–l) overexpression of vMIA-Myc in MFF silenced cells: (j) anti-TIM23, (k) anti-Myc, (l) merge image of j and k. Bars represent 10 µm. (B,C) Statistical analysis of mitochondrial morphologies upon overexpression of vMIA-Myc in MEFs MAVS-MITO cells in the absence of DLP1 or MFF, respectively. Approximately 600 cells were analysed per condition. Data represents the means ± SEM of three independent experiments analysed using two-way ANOVA with Bonferroni’s multi comparations test (ns = non-significant, ****– p < 0.0001). Error bars represent SEM.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Immunofluorescence, Control, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA-induced peroxisomal and mitochondrial fragmentation is independent of MAVS. vMIA does not disrupt STING-MAVS interaction at peroxisomes. (A) Immunofluorescence analyses of MEFs MAVS KO cells: (a, b) peroxisomal and mitochondrial morphologies in control cells: (a) anti-PMP70, (b) anti-TIM23; (c–e) peroxisomal morphology upon overexpression of vMIA-Myc: (c) anti-PMP70, (d) anti-Myc, (e) merge image of c and d; (f, h) mitochondrial morphology upon overexpression of vMIA-Myc: (f) anti-TIM23, (g) anti-Myc, (h) merge image of f and g. Bars represent 10 µm. (B,C) Statistical analysis of peroxisomal or mitochondrial morphologies upon overexpression of vMIA-Myc in MEFs MAVS KO cells, respectively. Approximately 600 cells were analysed per condition. (D) Co-immunoprecipitation analysis of the interaction between overexpressed STING-FLAG and vMIA-Myc in MEFs MAVS-PEX cells. The pull-down was performed using an antibody against MAVS. Western blot was performed with antibodies against FLAG and Myc. IN represents total cell lysate (input), IP represents immunoprecipitation and OUT represents the cell lysate extracted after incubation with the antibody (output). (E) Quantification of the ratio between IP and IN, in the presence or absence of vMIA. Data represents the means ± SEM of three independent experiments, analysed using unpaired T test (ns - non-significant; ***– p < 0.001, ****– p < 0.0001).

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Immunofluorescence, Control, Over Expression, Immunoprecipitation, Western Blot, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA inhibits MAVS oligomerization at peroxisomes and mitochondria. MFF is essential for the vMIA-mediated inhibition of MAVS oligomerization at peroxisomes but not at mitochondria. (A,B) HEK293T cells infected with SeV in the presence or absence of vMIA. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (A) Peroxisome-enriched fraction, (B) Mitochondria-enriched fraction. (C,D) HEK293T cells infected with SeV in the presence or absence of vMIA and in the absence of MFF. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (C) Peroxisome-enriched fraction, (D) Mitochondria-enriched fraction. (A–D) Immunoblots were performed with antibodies against MAVS, Myc-tag, COXIV, PEX14 and PMP70. (E) Whole cell lysates were resolved by SDS-PAGE. SeV infection, and consequential activation of MAVS downstream signalling, was confirmed using anti-p-STAT1. vMIA-Myc overexpression and MFF silencing were also confirmed using anti-Myc and anti-MFF, respectively. Antibody against Actin was used as loading control.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Inhibition, Infection, Isolation, Western Blot, SDS Page, Activation Assay, Over Expression, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Human myeloid dendritic cells (mDCs) were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells induced or not to realtime PCR. The profile of TRIM29 expression in different cells is indicated. The relative expression of TRIM29 was compared by plotting the values extracted from the gene expression database. A value < 1 indicated the absence of gene expression. (B) Immunoblot analysis of TRIM29 and MABVS in human mDCs treated with control shRNA with a scrambled sequence (sh-Ctrl), shRNA targeting mRNA encoding TRIM29 (two shRNAs: Trim29#1 and Trim29#2) or shRNA targeting mRNA encoding MAVS. GAPDH serves as a loading control throughout. (C) ELISA of IFN-β in human primary mDCs treated with scrambled shRNA (sh-Ctrl) and left unstimulated (Mock) or treated with shRNA as above and then stimulated for 16h with long poly I:C (LPIC, 20 μg/ml) delivered by Lipofectamine 3000. *P<0.05, **P<0.01, ***P<0.001 (unpaired t test). N-STM, cells without stimulation. Data are representative of three independent experiments with similar results (mean + s.d.).

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Purification, Isolation, Expressing, Gene Expression, Western Blot, Control, shRNA, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Immunoblot analysis of endogenous proteins of TRIM29 and MAVS precipitated with anti-MAVS, anti-TRIM29 or control immunoglobulin G (control IgG) from whole-cell lysates of BMDCs from wild type mice stimulated without (No) or with poly I:C (25 μg/ml) for 8h, and then with MG132 treatment for 3h. Input, 20% of the BMDCs lysate. (B) Full-length MAVS and serial truncations of MAVS with deletion of various domains (top). Below, immunoblot analysis of purified HA-tagged TRIM29 with anti-HA (bottom blot), and immunoblot analysis of purified Myc-tagged full-length MAVS and MAVS truncation mutants alone with anti-Myc (top blot) or after incubation with HA-tagged full-length TRIM29 and immunoprecipitation with anti-HA (middle blot). (C) Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains (left margin); numbers at ends indicate amino acid positions (top). Below, immunoblot analysis of purified Myc-tagged MAVS with anti-Myc (bottom blot), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone (top blot) or after incubation with Myc-tagged MAVS and immunoprecipitation with anti-Myc (middle blot). (D) Activation of the IFN-β promoter in human HEK293T cells transfected with an IFN-β promoter luciferase reporter, plus expression vector (each 100 ng) for wild-type MAVS or expression vector for wild-type TRIM29 (T29-a) or TRIM29 mutants T29-b and T29-g; results are presented relative to those of renilla luciferase (cotransfected as an internal control). (E) Colocalization of endogenous TRIM29 and MAVS in BMDCs. Confocal microscopy of BMDCs infected with reovirus for 4h. MAVS was stained with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (green), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue). Scale bars represent 20 μm. Data are representative of three independent experiments.

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Western Blot, Control, Purification, Incubation, Immunoprecipitation, Activation Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Confocal Microscopy, Infection, Staining, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Immunoblot analysis of Myc-tagged MAVS (top blot), HA-tagged TRIM29 (middle blot) and β-actin (bottom blot) in HEK293T cells cotransfected with an expression vector for Myc-tagged MAVS and empty vector or expression vector for HA-tagged TRIM29, with or without treatment of 5 μM MG132. (B) Immunoblot analysis of TRIM29 (top blot), MAVS (second blot), phosphorylated IRF3 (third blot), phosphorylated p65 (fourth blot) and β-actin (bottom blot) in WT and TRIM29 KO BMDCs stimulated for various times (above lanes) with poly I:C (10 μg/ml). (C) Immunoblot analysis (with anti-Myc) of the abundance (top), total ubiquitination (second blot) and K11-linked ubiquitination (third blot) of Myc-tagged MAVS in HEK293T cells transfected with empty vector or expression vector for HA-tagged TRIM29, truncation T29-g (losing binding site of MAVS), and stimulated for 4 h with poly I:C (20 μg/ml), assessed after immunoprecipitation with anti-Myc; and immunoblot analysis whole-cell lysates with anti-HA (fifth blot) and anti-β-actin (bottom). (D) Immunoblot analysis of TRIM29 in WT and KO BMDCs (top), and of the abundance (second blot), total ubiquitination (third blot) and K11-mediated ubiquitination (bottom blot) of MAVS in those cells, stimulated for 4 h with poly I:C (10 μg/ml), assessed after immunoprecipitation with anti-MAVS. Data are representative of three experiments.

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Western Blot, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Transfection, Binding Assay, Immunoprecipitation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Immunoblot analysis of Myc-tagged MAVS and its mutations (top blot), HA-tagged TRIM29 (middle blot) and β-actin (bottom blot) in HEK293T cells cotransfected with an expression vector for HA-tagged TRIM29 and expression vectors for Myc-tagged wild-type full-length MAVS and its mutations. (B) Immunoblot analysis of HA-tagged TRIM29 (top blot), Myc-tagged MAVS and its mutations (second blot), β-actin (third blot) and K11-linked ubiquitination (bottom blot) in HEK293T cells cotransfected with expression vector for Myc-tagged MAVS or its mutations (above lanes) and expression vector for HA-tagged TRIM29, with treatment of 5 μM MG132 (above lanes). (C,D) Activation of the IFN-β promoter (C) or NF-κB promoter (D) in human HEK293T cells transfected with an IFN-β promoter luciferase reporter (C) or NF-κB promoter luciferase reporter (D), plus expression vector (each 100 ng) for wild-type MAVS or various MAVS mutants alone or expression vector for TRIM29 plus wild-type MAVS or MAVS mutants; results are presented relative to those of renilla luciferase (cotransfected as an internal control). Data are representative of three independent experiments with similar results (mean + s.d.)

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Western Blot, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Activation Assay, Transfection, Luciferase, Control

Journal: Cells

Article Title: A Gain-of-Function Cleavage of TonEBP by Coronavirus NSP5 to Suppress IFN-β Expression.

doi: 10.3390/cells13191614

Figure Lengend Snippet: Figure 5. Cleavage of TonEBP promotes binding to the PRD II domain of the IFN-β promoter. (A) HEK293T cells were co-transfected for 16 h with a plasmid expressing GFP-IRF3 without or with the plasmid expressing Flag-TonEBP NT. Cell lysates (Input) were immunoprecipitated (IP) with anti- Flag antibody. (B) Cells were transfected and analyzed as in (A) except that YPF-p65 was expressed instead of GFP-IRF3. (C) HEK293T cells were co-transfected for 24 h with various combinations of two plasmids expressing Flag-TonEBP NT and MAVS, as indicated. Chromatin immunoprecipitation was performed using normal rabbit IgG or anti-TonEBP antibody. The IFN-β promoter region indicated was quantified using qPCR. Mean ± SEM, n = 3. (D) Cells were co-transfected for 24 h with various combinations of three plasmids expressing Flag-TonEBP, Flag-TonEBP Q1127A and SARS-CoV-2 NSP5, as indicated. Chromatin immunoprecipitation was performed as in (C). (E) Schematic of TonEBP NT (1–1127) and TonEBP sNT (1–548). (F) Cells were transfected for 24 h with the IFN- β promoter luciferase reporter plasmid and pRL-TK plus one of the combinations of plasmids expressing p65, TonEBP NT, and TonEBP sNT (1–548), as indicated. Luciferases were measured to assess the activity of IFN-β promoter. (G) Nucleotide sequence of a portion of the human IFN-β promoter for binding of IRF3 and NF-κB (p65). Sequences of three probes are shown below. PRDIII, PRDI, and PRDII regions are marked by yellow, green, and orange colors, respectively. (H,I) The probes shown above were biotinylated and individually immobilized on streptavidin sensor chips for surface plasmon resonance assay. Various concentrations of p65 (H) or TonEBP sNT (1–548) (I) recombinant proteins were injected over the sensor surface. Binding affinity was determined by plotting the maximum response unit versus the concentration of injected proteins using GraphPad Prism 9.0. The values of KD are shown. (J) 100 nM each of Cy5.5-labeled IFN-β promoter probes were individually incubated without or with 500 nM TonEBP sNT (1–548) and resolved in an agarose gel. (K) Combinations of 0, 100, 200, or 500 nM TonEBP sNT (1–548) and p65 recombinant proteins (0 or 100 nM), as indicated, were each incubated for 25 min with 100 nM of Cy5.5-labeled IRF3/p65 probe and resolved in an agarose gel. The solid arrowhead denotes the complex of TonEBP sNT (1–548) with the probe and the open arrowhead denotes the complex of p65 with the probe. (L) A model for the IFN-β induction and its inhibition by TonEBP NT by replacing DNA bound p65. Data are shown as the mean ± SEM, n = 3; ns p > 0.05, and *** p < 0.001 using t-tests (C,D,F).

Article Snippet: The IFN-β promoter luciferase reporter (#102597), as well as transient expression vectors for MDA5 (#52876), MAVS (#52135), TBK1 (#131792), IRF3 (#127663), and p65 (#111192), were obtained from Addgene.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Sequencing, SPR Assay, Recombinant, Injection, Concentration Assay, Labeling, Incubation, Agarose Gel Electrophoresis, Inhibition