Journal: The Journal of Biological Chemistry

Article Title: SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

doi: 10.1016/j.jbc.2024.105779

Figure Lengend Snippet: Nsp1 inhibited the IFN response. A–D , HEK-293T cells were cotransfected with pCAGGS-2×HA-STAT1/pCAGGS-2×HA-STAT2/pCAGGS-2×HA-IRF9/ISGF3 (pCAGGS-2×HA-STAT1, pCAGGS-2×HA-STAT2, and pCAGGS-2×HA-IRF9), pIFN-β-luc, and pRL-TK at a ratio of 1:0.2:0.05. After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1. After 24 h, the cells were collected to detect ISRE promoter activity through the dual luciferase reporter assay. E , HEK-293T cells were co-transfected with pCAGGS-3×Flag-nsp1, pIFN-β-luc, and pRL-TK at a ratio of 1:0.2:0.05. After 24 h, the cells were stimulated by human IFN-β for 4 h. Then, the cells were collected to detect ISRE promoter activity through the dual luciferase reporter assay. F , HEK-293T cells were transfected with pCAGGS-2×HA-STAT1/pCAGGS-2×HA-STAT2/pCAGGS-2×HA-IRF9/ISGF3 (pCAGGS-2×HA-STAT1, pCAGGS-2×HA-STAT2, and pCAGGS-2×HA-IRF9). After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1. The cells were then collected to detect the nsp1, STAT1, STAT2, IRF9, and ISGF3 expression levels via Western blotting. G–J , HEK-293T cells were transfected with pCAGGS-2×HA-STAT1/pCAGGS-2×HA-STAT2/pCAGGS-2×HA-IRF9/ISGF3 (pCAGGS-2×HA-STAT1, pCAGGS-2×HA-STAT2, and pCAGGS-2×HA-IRF9). After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1. Then, the cells were collected to detect the OAS1, ISG15, and ISG56 mRNA levels using RT-qPCR. J , HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1 or pCAGGS-3×Flag-nsp1-mutant. After 24 h, the cells were stimulated by SeV. Then, the cells were collected to detect the OAS1, ISG15, and ISG56 mRNA levels using RT-qPCR. Data are the mean ± SD. The p -value was calculated using the t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. HEK, human embryonic kidney cell line; IFN, interferon; IRF9, interferon regulatory factor-9; ISG, interferon-stimulated gene; ISGF3, interferon-stimulated gene factor 3; ISRE, interferon-stimulated response elements; nsp1, nonstructure protein 1; OAS1, 2′-5′-oligoadenylate synthetase 1; SeV, Sendai virus; STAT, signal transducer and activator of transcription.

Article Snippet: This study used the following antibodies and reagents: mouse anti-FLAG-tag monoclonal antibody (mAb), mouse anti-HA-tag mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse (H+L), HRP-conjugated goat anti-rabbit (H+L), HRP-conjugated mouse anti-rabbit (L), HRP-conjugated goat anti-mouse (L), p-STAT1 rabbit mAb, STAT1 rabbit mAb, p-STAT2 rabbit mAb, STAT2 rabbit mAb, IRF9 rabbit mAb, p-JAK1 rabbit mAb, JAK1 rabbit mAb, TYK2 rabbit mAb, and p-TYK2 rabbit mAb (ABclonal); CREB-BP rabbit mAb (Affinity); beta-actin rabbit antibody (Proteintech); MG132 and Z-VAD-FMK (Beyotime); CHX710 (MedChemExpress); human IFN-β (InvivoGen); Lipofectamine 3000 transfection reagent (Sigma); and Dylight-conjugated 488 goat anti-mouse IgG (Abbkine).

Techniques: Transfection, Activity Assay, Luciferase, Reporter Assay, Expressing, Western Blot, Quantitative RT-PCR, Mutagenesis, Virus

Journal: The Journal of Biological Chemistry

Article Title: SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

doi: 10.1016/j.jbc.2024.105779

Figure Lengend Snippet: Nsp1 inhibited STAT1 phosphorylation. A and B , HEK-293T cells and LLC-PK1 cells were transfected with pCAGGS-3×Flag-nsp1. After 24 h, HEK-293T cells were incubated with human IFN-β for 4 h, and LLC-PK1 cells were stimulated by SeV for 8 h. STAT1, p-STAT1, STAT2, and p-STAT2 expression was detected by Western blotting. All protein levels were analyzed using ImageJ. Western blotting assay was repeated in two independent experiments. C , HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1-mutant. After 24 h, HEK-293T cells were incubated with human IFN-β for 4 h. STAT1 and p-STAT1 expression was detected by Western blotting. All protein levels were analyzed using ImageJ. Western blotting assay was repeated in two independent experiments. The data are the means ± SD. The p -value was calculated using the t test. ∗∗∗ p < 0.001. HEK, human embryonic kidney cell line; IFN-β, interferon-β; nsp1, nonstructure protein 1; SeV, Sendai virus; STAT, signal transducer and activator of transcription.

Article Snippet: This study used the following antibodies and reagents: mouse anti-FLAG-tag monoclonal antibody (mAb), mouse anti-HA-tag mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse (H+L), HRP-conjugated goat anti-rabbit (H+L), HRP-conjugated mouse anti-rabbit (L), HRP-conjugated goat anti-mouse (L), p-STAT1 rabbit mAb, STAT1 rabbit mAb, p-STAT2 rabbit mAb, STAT2 rabbit mAb, IRF9 rabbit mAb, p-JAK1 rabbit mAb, JAK1 rabbit mAb, TYK2 rabbit mAb, and p-TYK2 rabbit mAb (ABclonal); CREB-BP rabbit mAb (Affinity); beta-actin rabbit antibody (Proteintech); MG132 and Z-VAD-FMK (Beyotime); CHX710 (MedChemExpress); human IFN-β (InvivoGen); Lipofectamine 3000 transfection reagent (Sigma); and Dylight-conjugated 488 goat anti-mouse IgG (Abbkine).

Techniques: Phospho-proteomics, Transfection, Incubation, Expressing, Western Blot, Mutagenesis, Virus

Journal: The Journal of Biological Chemistry

Article Title: The main protease of SARS-CoV-2 cleaves histone deacetylases and DCP1A, attenuating the immune defense of the interferon-stimulated genes

doi: 10.1016/j.jbc.2023.102990

Figure Lengend Snippet: M pro shows no interaction with IFN signaling pathway transducers. (A) HEK-293T cells were transfected with a C-terminal flag-tagged EGFP-N15/N16-BFP plasmid along with SARS-CoV-2 M pro or empty vector. Cells were lysed at 24 h after transfection and analyzed by western blotting. (B) HEK-293T cells were co-transfected with a C-terminal flag-tagged STAT1, STAT2, IRF9, JAK1, or TYK2 expression plasmid combined either with SARS-CoV-2 M pro or empty vector. Whole-cell extracts were lysed 30 h post-transfection and analyzed by western blotting. (C) HeLa cells were transfected with pCDNA3.1-M pro -mCherry visual construct, 24 h post-transfection, cells were treated with or without IFNα (1000 U/mL) for 1 h and possessed for indirect immunofluorescence to detect the STAT1, STAT2, and IRF9. Scale bar, 10 μm. (D) HEK-293T cells were transfected with SARS-CoV-2 M pro or empty vector. After 24 h of expressing, cells were cultured with or without IFNα (1000 U/mL) for 1 h. whole-cell extracts or the nuclear and cytoplasmic fractions were analyzed by western blotting with specific antibodies for the detection of tyrosine phospho-STAT1 and STAT2 or total STAT1 and STAT2. (E) Density analysis represents the relative protein levels of phospho-STAT1 or phospho-STAT2 that was normalized to the protein levels of α-tubulin or PCNA. The value of control group was set to 1. The presented results represent the means and standard deviations of data from three independent experiments. Statistical significance was calculated using unpaired, two-tailed Student’s t test. Protein band intensities were quantitated by Image Lab software.

Article Snippet: The following antibodies were used: Mouse (Ms) anti-FLAG (Sigma-Aldrich, F1804), Ms anti-HA (Cell Signaling, 2367), Ms anti-HA (Biolegend,901501), Ms HRP-conjugated anti-Alpha Tubulin (Proteintech, HRP-66031), Ms HRP-conjugated anti-GAPDH (Yesen, 30203ES50), Rabbit (Rb) anti-STAT1 (Cell Signaling, 14994), Rb anti-STAT2 (Proteintech, 16674-1-AP), Ms anti-p-STAT1 (Santa Cruz, sc-136229), Rb anti-p-STAT2 (Cell Signaling, 4441), and Ms anti-IRF9 (Santa Cruz, sc-365893).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Construct, Immunofluorescence, Cell Culture, Two Tailed Test, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) and Spike (S) Proteins Antagonize Host Type I Interferon Response

doi: 10.3389/fcimb.2021.766922

Figure Lengend Snippet: M protein inhibits the interaction between KPNA6 and IRF3. (A) Partial results of the IP-MS assay using Flag-tagged M protein as a bait. HEK293T cells were co-transfected with plasmid encoding Flag-tagged SARS-CoV-2 M protein. At 24 hpt., cells were treated with SeV for 12 h Co-IP was performed by incubating the cell lysates with anti-Flag magnetic beads overnight, and eluted proteins were subjected to western blotting verification to confirm successful IP of viral proteins. The elution mixture was processed for protein identification by Mass Spectrometry. The results were analyzed by Proteome Discoverer 2.2 software. (B) Co-IP of M and IPed KPNA proteins. HEK293T cells were co-transfected with Flag-tagged SARS-CoV-2 M plasmid and each Myc-tagged KPNA2/KPNA3/KPNA4/KPNA6 plasmid. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-Flag antibody magnetic beads overnight. After extensive washing, the eluate was analyzed by western blotting with indicated antibodies. (C) Co-IP of IRF3 and KPNA2 or KPNA6. HEK293T cells were co-transfected with HA-tagged IRF3 plasmids and Myc-tagged KPNA2 or KPNA6 plasmids or empty plasmids. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-HA antibody overnight before the addition of magnetic beads. (D) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA2-Myc, together with or without M-Flag. The cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (E) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA6-Myc, together with or without M-Flag. Then the cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (F) Interaction between endogenous IRF3 and KPNA6 in the presence of M protein. HEK293T cells were transfected with M-Flag for 24 h and then treated with SeV for another 12 h, the cells were harvested and subjected to a Co-IP assay using IgG or IRF3 antibody. (G) HEK293T cells were transfected with M-Flag for 24 h and treated with SeV for 12 h. IRF3 in the nuclear fractions or the cytoplasmic was determined by immunoblotting analyses. GAPDH and Lamin B1 served as cytoplasmic and nuclear protein controls, respectively. (H) Nuclear translocation of IRF3. HeLa cells were transfected with M-Flag or empty vector. At 24 hpt., cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with PBS containing 2% fetal bovine serum (FBS), the cells were probed with primary antibodies (anti-Flag and anti-IRF3) and secondary antibodies (anti-Alexa Fluor 488 and anti-Alexa Fluor 648). Scale bar, 10 µm. Representative blots and fluorescence pictures of 3 independent experiments were shown. The percentages of IRF3 in the nucleus (out of total IRF3 signal) were quantified by ImageJ software. ***P<0.001, t -test.

Article Snippet: The following antibodies were used: rabbit anti-DYKDDDDK tag (ABclonal, Cat # AE005), rabbit anti-HA (CST, Cat # 3724S), rabbit anti-IRF3 (ABclonal, Cat # D199862-0100), rabbit anti-p-IRF3 (CST, Cat # 4947S), rabbit anti-TBK1 (4A Biotech co.Ltd, Cat# 4ab032308cs), rabbit anti-p-TBK1 (Absin, Cat # abs140019), rabbit anti-KPNA6 (ImmunoWay, Cat # YN3049), rabbit anti-STAT2 (CST, Cat # 72604S), rabbit anti-p-STAT2 (CST, Cat # 88410S), rabbit anti-p-STAT1 (CST, Cat # 9167S), rabbit anti-p-TYK2 (Absin, Cat # abs131318), rabbit anti-TYK2 (Absin, Cat # abs131318a), rabbit anti-Myc (Proteintech, Cat # I6286-I-AP), anti-rabbit IgG HRP-linked antibody (CST, Cat # 7074), mouse anti-DYKDDDDK tag (Bioworld, Cat # AP0007), mouse anti-GAPDH (Proteintech, Cat # 60004-I-Ig), mouse anti-VSV-G (Abgent, Cat # AP1016a) and HRP goat anti-mouse IgG (BioLegend, Cat # 405306).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Mass Spectrometry, Software, Translocation Assay, Blocking Assay, Fluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) and Spike (S) Proteins Antagonize Host Type I Interferon Response

doi: 10.3389/fcimb.2021.766922

Figure Lengend Snippet: A subset of viral proteins antagonize IFN-β production. (A) HEK293T cells cultured in 24-well plates (1 × 10 5 cells per well) were transfected with Flag-N1 empty vector (200 ng) or the SARS-CoV-2 protein plasmids (200 ng). At 24 h after transfection, cells were stimulated by SeV (MOI=1), and at 12 h after stimulation, the cells were harvested for RNA extraction and subsequent RT-qPCR analysis to assess the expression of IFN-β or ISG (IFIT1). (B) The Flag-N1 empty vector and the SARS-CoV-2 protein plasmids (100 ng) were transfected with the indicated combinations of plasmids expressing RIG-IN (10 ng), MAVS (10 ng), TBK1 (100 ng), and IRF3-5D (10 ng) into HEK293T cells cultured in 96-well plates (0.5 × 10 5 cells per well). The IFN-β-Luc (50 ng) plasmids were co-transfected to assess the activation of IFN promoter and the pRL-TK (5 ng) was transfected as an internal control. Dual luciferase assays were performed 36 hpt. Results were shown as Mean ± SD. Statistical significance was assessed via comparison to the Flag-N1 control using one-way ANOVA with Dunnett’s correction, **p < 0.01, ***p < 0.001. ns, not significant. The data shown are representative of 3 independent experiments. (C) Phosphorylation of IRF3 and TBK1. HEK293T cells were transfected with viral protein-encoding plasmids (1 µg), treated with SeV for 12 h, and analyzed for phosphorylated IRF3 (anti-p-IRF3 at S396), total IRF3 (anti-IRF3), phosphorylated TBK1 (anti-p-TBK1 at S172), total TBK1 (anti-TBK1), and GAPDH (anti-GAPDH) by western blotting. Representative blots of three independent experiments are shown. (D) Summary of the antagonism of IFN-I production. The potential inhibitory steps are indicated for individual viral proteins.

Article Snippet: The following antibodies were used: rabbit anti-DYKDDDDK tag (ABclonal, Cat # AE005), rabbit anti-HA (CST, Cat # 3724S), rabbit anti-IRF3 (ABclonal, Cat # D199862-0100), rabbit anti-p-IRF3 (CST, Cat # 4947S), rabbit anti-TBK1 (4A Biotech co.Ltd, Cat# 4ab032308cs), rabbit anti-p-TBK1 (Absin, Cat # abs140019), rabbit anti-KPNA6 (ImmunoWay, Cat # YN3049), rabbit anti-STAT2 (CST, Cat # 72604S), rabbit anti-p-STAT2 (CST, Cat # 88410S), rabbit anti-p-STAT1 (CST, Cat # 9167S), rabbit anti-p-TYK2 (Absin, Cat # abs131318), rabbit anti-TYK2 (Absin, Cat # abs131318a), rabbit anti-Myc (Proteintech, Cat # I6286-I-AP), anti-rabbit IgG HRP-linked antibody (CST, Cat # 7074), mouse anti-DYKDDDDK tag (Bioworld, Cat # AP0007), mouse anti-GAPDH (Proteintech, Cat # 60004-I-Ig), mouse anti-VSV-G (Abgent, Cat # AP1016a) and HRP goat anti-mouse IgG (BioLegend, Cat # 405306).

Techniques: Cell Culture, Transfection, Plasmid Preparation, RNA Extraction, Quantitative RT-PCR, Expressing, Activation Assay, Luciferase, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) and Spike (S) Proteins Antagonize Host Type I Interferon Response

doi: 10.3389/fcimb.2021.766922

Figure Lengend Snippet: M protein inhibits the interaction between KPNA6 and IRF3. (A) Partial results of the IP-MS assay using Flag-tagged M protein as a bait. HEK293T cells were co-transfected with plasmid encoding Flag-tagged SARS-CoV-2 M protein. At 24 hpt., cells were treated with SeV for 12 h Co-IP was performed by incubating the cell lysates with anti-Flag magnetic beads overnight, and eluted proteins were subjected to western blotting verification to confirm successful IP of viral proteins. The elution mixture was processed for protein identification by Mass Spectrometry. The results were analyzed by Proteome Discoverer 2.2 software. (B) Co-IP of M and IPed KPNA proteins. HEK293T cells were co-transfected with Flag-tagged SARS-CoV-2 M plasmid and each Myc-tagged KPNA2/KPNA3/KPNA4/KPNA6 plasmid. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-Flag antibody magnetic beads overnight. After extensive washing, the eluate was analyzed by western blotting with indicated antibodies. (C) Co-IP of IRF3 and KPNA2 or KPNA6. HEK293T cells were co-transfected with HA-tagged IRF3 plasmids and Myc-tagged KPNA2 or KPNA6 plasmids or empty plasmids. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-HA antibody overnight before the addition of magnetic beads. (D) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA2-Myc, together with or without M-Flag. The cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (E) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA6-Myc, together with or without M-Flag. Then the cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (F) Interaction between endogenous IRF3 and KPNA6 in the presence of M protein. HEK293T cells were transfected with M-Flag for 24 h and then treated with SeV for another 12 h, the cells were harvested and subjected to a Co-IP assay using IgG or IRF3 antibody. (G) HEK293T cells were transfected with M-Flag for 24 h and treated with SeV for 12 h. IRF3 in the nuclear fractions or the cytoplasmic was determined by immunoblotting analyses. GAPDH and Lamin B1 served as cytoplasmic and nuclear protein controls, respectively. (H) Nuclear translocation of IRF3. HeLa cells were transfected with M-Flag or empty vector. At 24 hpt., cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with PBS containing 2% fetal bovine serum (FBS), the cells were probed with primary antibodies (anti-Flag and anti-IRF3) and secondary antibodies (anti-Alexa Fluor 488 and anti-Alexa Fluor 648). Scale bar, 10 µm. Representative blots and fluorescence pictures of 3 independent experiments were shown. The percentages of IRF3 in the nucleus (out of total IRF3 signal) were quantified by ImageJ software. ***P<0.001, t -test.

Article Snippet: The following antibodies were used: rabbit anti-DYKDDDDK tag (ABclonal, Cat # AE005), rabbit anti-HA (CST, Cat # 3724S), rabbit anti-IRF3 (ABclonal, Cat # D199862-0100), rabbit anti-p-IRF3 (CST, Cat # 4947S), rabbit anti-TBK1 (4A Biotech co.Ltd, Cat# 4ab032308cs), rabbit anti-p-TBK1 (Absin, Cat # abs140019), rabbit anti-KPNA6 (ImmunoWay, Cat # YN3049), rabbit anti-STAT2 (CST, Cat # 72604S), rabbit anti-p-STAT2 (CST, Cat # 88410S), rabbit anti-p-STAT1 (CST, Cat # 9167S), rabbit anti-p-TYK2 (Absin, Cat # abs131318), rabbit anti-TYK2 (Absin, Cat # abs131318a), rabbit anti-Myc (Proteintech, Cat # I6286-I-AP), anti-rabbit IgG HRP-linked antibody (CST, Cat # 7074), mouse anti-DYKDDDDK tag (Bioworld, Cat # AP0007), mouse anti-GAPDH (Proteintech, Cat # 60004-I-Ig), mouse anti-VSV-G (Abgent, Cat # AP1016a) and HRP goat anti-mouse IgG (BioLegend, Cat # 405306).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Mass Spectrometry, Software, Translocation Assay, Blocking Assay, Fluorescence