Journal: eBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: BTN3A2 was identified as a potent restriction factor for SARS-CoV-2 infection . a : Overexpression of BTN3A family members inhibited SARS-CoV-2 replication in A549 cells overexpressing human ACE2 (ACE2-A549). ACE2-A549 cells (1 × 10 5 ) were transfected with indicated expression vector (BTN3A2-L, BTN3A2-S, BTN3A1, or BTN3A3) or empty vector (Vector) (each 1 μg) for 24 h, then infected with SARS-CoV-2 (MOI = 0.01) for 24 h. Cells were harvested to determine SARS-CoV-2 copies by qRT-PCR. b : Overexpression of BTN3A2 inhibited SARS-CoV-2 replication in HEK293T cells overexpressing human ACE2 (ACE2-HEK293T). ACE2-HEK293T cells (5 × 10 4 ) were transfected with indicated expression vector (BTN3A2-L or BTN3A2-S) or empty vector (each 1 μg) for 24 h, then infected with SARS-CoV-2 (MOI = 0.01 or 0.001) for 24 h and harvested for quantification of SARS-CoV-2 copies. c : Stable expression of BTN3A2 in Huh7 cells inhibited SARS-CoV-2 replication. Huh7 cells overexpressing BTN3A2-L or BTN3A2-S, and control cells (Vector Huh7) (1 × 10 5 cells) were treated with DOX (1 μg/mL) for 24 h, then challenged with the SARS-CoV-2 prototype or BA.2 (MOI = 1) for 24 h. The culture medium (supernatant, left ) and cell lysates (cytoplasm, right ) were collected to quantify SARS-CoV-2 copies. d : Stable expression of BTN3A2 in Calu-3 cells inhibited replication of VSV pseudotyped SARS-CoV-2 (prototype and BA.1). The procedure was similar to that in ( c ). After 24 h infection, cells were collected, and viral infectivity was quantified based on Renilla luciferase activity (RLU) using a Renilla Luciferase Assay Kit (Promega, Madison, WI, USA). e : Incubation with BTN3A2 isoforms (BTN3A2-L-Fc and BTN3A2-S-His) inhibited replication of VSV pseudotyped SARS-CoV-2 (BA.1) in Calu-3 cells. IgG treatment was used as a negative control. Cells were incubated with BTN3A2 isoform or IgG for 1 h, then infected with VSV pseudotyped SARS-CoV-2 (BA.1) for 24 h and harvested to determine viral infectivity. f : Knockout of BTN3A2 increased SARS-CoV-2 replication in Huh7 cells. Huh7 cells with BTN3A2 knockout (sgBTN3A2-1 and sgBTN3A2-3 Huh7) and control cells (sgNC Huh7) were infected with SARS-CoV-2 prototype or BA.2 (MOI = 1) for 24 h, then harvested to quantify SARS-CoV-2 copies. g : Knockout of BTN3A2 increased VSV pseudotyped SARS-CoV-2 (prototype and BA.1) replication in Calu-3 cells. The procedure for determining viral infectivity was the same as that in ( d ). Data shown in ( b-d ) were average quantitative data from three independent experiments and were presented as mean ± standard deviation (SD). Significance was determined by comparing to Vector in each group (a, b, c, and d), IgG control at each dilution (e), or sgNC in each group (f and g). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons.

Article Snippet: To measure the cell surface expression level of ACE2, Huh7 cells and Calu-3 cells with BTN3A2 overexpression (BTN3A2-L Huh7 cells, BTN3A2-S Huh7 cells, BTN3A2-L Calu-3 cells and BTN3A2-S Calu-3 cells) or knockout (sgBTN3A2-1 Huh7 cells, sgBTN3A2-3 Huh7 cells, sgBTN3A2-1 Calu-3 cells, and sgBTN3A2-3 Calu-3 cells), and corresponding control cells (Vector Huh7, Vector Calu-3, sgNC Huh7, and sgNC Calu-3) were washed with PBS and stained with mouse anti-ACE2 (66699-1-Ig, Proteintech, China) at a 1:50 dilution for 30 min at 4 °C.

Techniques: Infection, Over Expression, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Control, Luciferase, Activity Assay, Incubation, Negative Control, Knock-Out, Standard Deviation

Journal: eBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: BTN3A2 inhibited SARS-CoV-2 entry and interacted with Spike protein via its RBD . a : Stable expression of BTN3A2 suppressed SARS-CoV-2 attachment to the cell. Huh7 cells overexpressing BTN3A2-L (BTN3A2-L Huh7) or BTN3A2-S (BTN3A2-S Huh7) and the control (Vector Huh7) cells were treated with DOX (1 μg/mL) for 24 h, then incubated with SARS-CoV-2 (MOI = 1) on ice at 4 °C for 1 h. Cells were washed by PBS three times and harvested to measure viral RNA copies based on SARS-CoV-2 N gene level. b : Representative immunofluorescence images of cell–cell fusion assay showing no apparent effect of BTN3A2 overexpression (BTN3A2-L and BTN3A2-S) on syncytia formation mediated by Spike of Delta. The HEK293T cells were transfected with Spike of Delta expression vector, and the Huh7 cells were transfected with px-300 for 16 h. The transfected Huh7 and HEK293T cells were co-cultured for 20 h and harvested for immunofluorescence assays. Scale bar, 10 μm. c : BTN3A2 interacted with the SARS-CoV-2 Spike protein. HEK293T cells (1 × 10 7 ) were co-transfected with expression vector of BTN3A2 isoform (BTN3A2-L-His or BTN3A2-S-His, each 5 μg) and Spike protein (5 μg) for 48 h. Whole-cell lysates were immunoprecipitated (IP) using anti-His antibody. Immunoblotting (IB) for His (BTN3A2-L-His or BTN3A2-S-His), Spike, and Tubulin was performed using anti-His, anti-Spike, and anti-Tubulin antibodies, respectively. d : BTN3A2-L interacted with the S1 subunit but not the S2 subunit of Spike protein. Spike S1 and S2 subunits were Flag-tagged. e : Endogenous BTN3A2 interacted with the Spike S1 protein. Huh7 cells (1 × 10 8 ) were transfected with expression vector for Spike S1 protein (S1-Flag, 20 μg) or empty vector (Vector) for 36 h, then cells were treated with or without IFN-α (450 U) for 12 h. Whole-cell lysates were immunoprecipitated (IP) using anti-Flag antibody. Immunoblotting (IB) for BTN3A2, Flag (S1-Flag), and Tubulin was performed using anti- BTN3A2, anti-Flag, and anti-Tubulin antibodies, respectively. f-g : Biolayer interferometry analyses of Spike S1 protein binding to immobilized BTN3A2-L-His ( f ) and BTN3A2-S-His ( g ). Different concentrations (1 000, 500, 250, and 125 nM, corresponding to kinetic curves from top to bottom) of Spike S1 protein were used for binding to BTN3A2-L-His and BTN3A2-S-His (lacked 125 nM), respectively. h : BTN3A2-S interacted with the Flag-tagged S1 subunit of the Spike protein. i : BTN3A2 interacted with Spike RBDs of different SARS-CoV-2 variants. Procedures for IP and IB in ( d ), ( h ) and ( i ) are the same as those in ( c ). Data are representative of three independent experiments with similar results ( b – i ). ( a ) Values were presented as mean ± SD, and differences between groups were determined by comparing to the Dox-group; ANOVA with Dunnett's multiple comparisons, ∗∗∗, P < 0.001.

Article Snippet: To measure the cell surface expression level of ACE2, Huh7 cells and Calu-3 cells with BTN3A2 overexpression (BTN3A2-L Huh7 cells, BTN3A2-S Huh7 cells, BTN3A2-L Calu-3 cells and BTN3A2-S Calu-3 cells) or knockout (sgBTN3A2-1 Huh7 cells, sgBTN3A2-3 Huh7 cells, sgBTN3A2-1 Calu-3 cells, and sgBTN3A2-3 Calu-3 cells), and corresponding control cells (Vector Huh7, Vector Calu-3, sgNC Huh7, and sgNC Calu-3) were washed with PBS and stained with mouse anti-ACE2 (66699-1-Ig, Proteintech, China) at a 1:50 dilution for 30 min at 4 °C.

Techniques: Expressing, Control, Plasmid Preparation, Incubation, Immunofluorescence, Cell-Cell Fusion Assay, Over Expression, Transfection, Cell Culture, Immunoprecipitation, Western Blot, Protein Binding, Binding Assay

Journal: eBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: BTN3A2 inhibited ACE2 expression . a : Differential expression of BTN3A2 and ACE2 in scRNA-seq dataset of lung tissues from COVID-19 patients and uninfected individuals. Original dataset was reported in Melms et al. Significance was assessed by two-sided Wilcox rank sum test. b : Stable expression of BTN3A2 decreased ACE2 mRNA level in Huh7 cells. RNA was extracted from Huh7 cells overexpressing BTN3A2-L and BTN3A2-S, or control (Vector Huh7) cells (each group 1 × 10 5 cells). BTN3A2 and ACE2 mRNA levels were measured by qRT-PCR, with normalization to GAPDH . c-d : Overexpression of BTN3A2-L ( c ) and BTN3A2-S ( d ) suppressed ACE2 protein expression in HEK293T cells. Cells (5 × 10 5 ) were transfected with indicated expression vector or empty vector (each 1.25 μg) for 48 h, then harvested for western blotting. e : Dose-dependent inhibitory effect of BTN3A2-L on ACE2 protein expression. HEK293T cells (5 × 10 5 ) were transfected with indicated expression vector or empty vector (each 0.5 μg), together with increased BTN3A2-L-His (0.25, 0.50, 0.75, and 1.50 μg, with empty vector to reach a total amount of 2.5 μg of plasmid) for 48 h, then harvested for western blotting. f : Knockout of BTN3A2 increased ACE2 mRNA level in Huh7 cells. The procedure was similar to that in (b). g : Knockout of BTN3A2 promoted ACE2 protein expression in Huh7 ( upper ) and Calu-3 ( lower ) cells. h-i : Flow cytometry was conducted to show surface ACE2 in Calu-3 cells with BTN3A2 overexpression (BTN3A2-L and BTN3A2-S) or knockout (sgBTN3A2-1 and sgBTN3A2-2), together with the control cells (vector and sgNC) and unstained cells. Cell surface expression of ACE2 was measured ( h ), and quantification of mean fluorescence intensity (MFI (%), n = 3) was based on three independent experiments ( i ). j : ACE2 in BTN3A2-tg and WT mice infected with indicated AAV-GFP and AAV-hACE2. Immunoblotting for ACE2, BTN3A2, GFP, and Tubulin was performed using anti-ACE2, anti-BTN3A2, anti-GFP, and anti-Tubulin antibodies, respectively. k : Representative images of immunohistochemical staining of ACE2 and BTN3A2 in lung tissues in ( j ). Scale bar, 100 μm. Data were representative of three independent experiments with similar results ( b-k ). Values were presented as mean ± SD ( b , f , and i ). Significance was determined compared to Vector ( b and i ) or sgNC ( f and i ). ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ANOVA with Dunnett's multiple comparisons.

Article Snippet: To measure the cell surface expression level of ACE2, Huh7 cells and Calu-3 cells with BTN3A2 overexpression (BTN3A2-L Huh7 cells, BTN3A2-S Huh7 cells, BTN3A2-L Calu-3 cells and BTN3A2-S Calu-3 cells) or knockout (sgBTN3A2-1 Huh7 cells, sgBTN3A2-3 Huh7 cells, sgBTN3A2-1 Calu-3 cells, and sgBTN3A2-3 Calu-3 cells), and corresponding control cells (Vector Huh7, Vector Calu-3, sgNC Huh7, and sgNC Calu-3) were washed with PBS and stained with mouse anti-ACE2 (66699-1-Ig, Proteintech, China) at a 1:50 dilution for 30 min at 4 °C.

Techniques: Expressing, Control, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Transfection, Western Blot, Knock-Out, Flow Cytometry, Fluorescence, Infection, Immunohistochemical staining, Staining

Journal: Clinical and Experimental Immunology

Article Title: Association of immunoglobulin G4 and free light chain with idiopathic pleural effusion

doi: 10.1111/cei.12999

Figure Lengend Snippet: Analysis of the clonality of the effusion immunoglobulin (Ig)G4 antibodies of patients in the IgG4+ group by two‐dimensional electrophoresis (2‐DE). Control IgG4 κ myeloma protein from Sigma (cat no. I4639) (a –c). Effusion IgG4 antibodies of representative cases with abnormal IgG4 levels (d–i). The H and L chains were probed with anti‐IgG4‐Fc (left), anti‐κ chain (middle) and anti‐λ chain (right) antibodies.

Article Snippet: IEF was run in Mini‐PROTEAN 2‐D Electrophoresis Cell (Bio‐Rad) at 200 V for 15 min, 400 V for 15 min and 750 V for 2·5 h. After IEF, capillary gel was equilibrated with sodium dodecyl sulphate (SDS) sample buffer containing 5% β‐mercaptoethanol for 30 min. After washing the capillary gel with SDS running buffer, SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) was performed at 25 mA constant per gel.

Techniques: Electrophoresis