Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation drives plasma membrane accumulation of NS3/NS3A. ( A ) The stability of NS3/NS3A WT and NS3/NS3A N150Q proteins was examined in both transfected (top panels) and BTV-20-infected cells (bottom panels; MOI = 10). For transfection assays, HEK-293T cells were transfected with plasmids expressing NS3/NS3A WT or the N150Q mutant and treated with cycloheximide (CHX; 100 μg/mL) at 18 h post-transfection (designated as 0 h post-CHX treatment) to block de novo protein synthesis. For infection assays, MDOK cells were infected with BTV-20 WT or BTV-20 N150Q and treated with CHX (100 μg/mL) at 10 h post-infection (designated as 0 h post-CHX treatment). Cells were harvested at the indicated time points and analyzed by Western blotting. ( B ) Quantification of NS3/NS3A protein levels shown in panel A was performed by ImageJ densitometric analysis. Protein levels at each time point were normalized to the corresponding 0 h post-CHX treatment (18 h post-transfection or 10 h post-infection, respectively). ( C ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with ER marker anti-calnexin (green) or Golgi marker anti-syntaxin 6 (green). Fluorescence distribution was evaluated using line-scan intensity profiles. Scale bar, 5 µm. ( D ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with plasma membrane marker WGA-Alexa Fluor 488 (green). Line-scan intensity profiles are shown. Scale bar, 5 µm. ( E ) Plasma membrane isolation of HEK-293T cells transfected with NS3/NS3A WT or N150Q mutant, followed by Western blot analysis. PM (plasma membrane fraction); NPM (non-plasma membrane fraction); Total (plasma membrane fraction + non-plasma membrane fraction). ( F ) Quantification of NS3/NS3A at the plasma membrane fraction was analyzed by Image J from panel E (* P < 0.05, two-tailed unpaired t-test). ( G ) Confocal imaging of HeLa cells co-transfected with NS3/NS3A (WT or N150Q, red) and VP2 (green) or VP5 (green), showing their subcellular colocalization. Colocalization was assessed by line-scan intensity profiles.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Transfection, Infection, Expressing, Mutagenesis, Blocking Assay, Western Blot, Staining, Marker, Fluorescence, Isolation, Two Tailed Test, Imaging

Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation of NS3/NS3A facilitates its raft-enriched membrane association and efficient BTV release. ( A ) Representative confocal images of HeLa cells transfected with NS3/NS3A WT or the N150Q mutant. Cells were fixed and stained 20 h post-transfection. NS3/NS3A was detected using anti-NS3/NS3A (red), and lipid raft-enriched membrane domains were labeled with anti-flotillin-1 (green). Scale bars, 5 μm. ( B and C ) Confocal images of HeLa cells expressing NS3/NS3A treated with trifluoperazine (TFP) ( B ) or methyl-β-cyclodextrin (MβCD) ( C ). Cells were fixed and stained 20 h post-transfection. NS3/NS3A is shown in red and flotillin-1 in green. Fluorescence intensity profiles were analyzed along the indicated line scans. Scale bars, 5 μm. ( D ) Localization of NS3/NS3A at the plasma membrane of HeLa cells with or without MβCD or TFP treatment. The plasma membrane was stained using WGA-Alexa Fluor 488 (green), and NS3/NS3A was visualized in red. Scale bars, 5 μm. ( E ) Quantification of plasma membrane (PM) localization from panel D, shown as the ratio of PM-associated NS3/NS3A fluorescence intensity (defined by WGA staining) to total cellular NS3/NS3A intensity. Values were normalized to vehicle-treated controls and are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (** P < 0.01, **** P < 0.0001). ( F ) MDOK or HEK-293T cells were infected with BTV-20 WT or BTV-20 N150Q at an MOI of 5. At 6 h post-infection, the cells were treated with MβCD or TFP, and viral titers were determined at 12 h post-infection. Data represent mean ± SD ( n = 3 biological replicates). Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; *** P < 0.001; **** P < 0.0001). ( G ) Analysis of BTV-20 release efficiency following raft disruption. MDOK cells were infected with BTV-20 (MOI = 5) and then treated with MβCD or TFP. Extracellular and intracellular viral titers were determined at the indicated time points. Release efficiency was calculated as [extracellular titer/(extracellular + intracellular titer)] × 100%. Data are presented as mean ± SD ( n = 3 biological replicates). Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). ( H ) A total of 1,136 host proteins were identified by quantitative proteomics, of which 102 proteins were enriched in the NS3/NS3A WT interactome relative to the N150Q mutant (log₂ FC > 4 or unique to WT). Highlighted are the top eight candidates. ( I ) Gene Ontology (GO) enrichment analysis of proteins enriched in the NS3/NS3A WT interactome. Significantly overrepresented terms are shown for the biological process, cellular component, and molecular function categories. Bars represent −log₁₀ ( P value). ( J ) Sucrose gradient fractionation followed by immunoblotting of lysates from transfected HEK-293T cells, or from infected MDOK cells. DRM (raft-enriched) fractions correspond to fractions 5–12, whereas detergent-soluble membrane (DSM) fractions correspond to fractions 13–20. ( K ) Co-immunoprecipitation analysis of NS3/NS3A WT , NS3/NS3A N150Q , and FLNA. Cells were transfected with NS3/NS3A variants, followed by Co-IP.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Membrane, Transfection, Mutagenesis, Staining, Labeling, Expressing, Fluorescence, Clinical Proteomics, Infection, Disruption, Quantitative Proteomics, Fractionation, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay