nxf1  (Bethyl)

Journal: Science Advances

Article Title: Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression

doi: 10.1126/sciadv.abe7386

Figure Lengend Snippet: ( A ) Schematic representation of NXF1-NXT1 and Nsp1. NXF1 contains an N-terminal RNA recognition motif (RRM), a leucine-rich repeat domain (LRR), a nuclear transport factor 2-like domain (NTF2L), and a ubiquitin-associated domain (UBA). RRM and LRR domains form an RNA binding module. NXT1 forms a heterodimer with NXF1 by binding to the NTF2L domain of NXF1. Nsp1 contains a β barrel domain. ( B ) 293T cells were transfected with 3xFlag-Nsp1 for 24 hours and then subjected to fractionation into nuclear and cytoplasmic fractions. See fig. S1 for fractionation controls. Nuclear lysates were subjected to immunoprecipitation (IP), followed by Western blot analysis to detect the indicated proteins. n = 3. ( C ) 293T cells expressing the SARS-CoV-2 receptor ACE2 protein were infected with SARS-CoV-2 at an MOI of 1 for 24 hours. Nsp1 was specifically immunoprecipitated from the cell lysates, and NXF1 interaction was detected by Western blot analysis. n = 3. ( D ) In vitro GST pull-down assays using the depicted purified recombinant proteins show that Nsp1 directly binds to NXF1. n = 3. ( E ) NXF1 associates with RNA in the presence of Nsp1. An electrophoretic mobility assay was carried out with a fluorescently labeled poly(U) 15-mer RNA and purified recombinant NXF1 (RRM-LRR) and/or Nsp1 (1-129) as indicated. n = 3.

Article Snippet: Serum containing anti-Tpr antibodies was used at 1:200 dilution, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (GeneTex, GTX627408) was used at 1:10,000 dilution; monoclonal mouse anti-NXF1 antibody was used at 1:1000 dilution (Sigma-Aldrich, T1076); polyclonal rabbit anti-NXF1 antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-915A); polyclonal rabbit anti-GANP antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-128A); polyclonal rabbit anti-THOC3 antibody was used at 1:1000 dilution (Bethyl Laboratories, A304-870A); polyclonal rabbit anti-nucleolin antibody was used at 1:1000 dilution (Novus Biologicals, NB600-241); polyclonal rabbit anti-UAP56 antibody was used at 1:1000 dilution (Sigma-Aldrich, SAB1307254); polyclonal rabbit anti-hnRNP K antibody was used at 1:2500 dilution (GeneTex, GTX101786); monoclonal rabbit anti-tubulin antibody was used at 1:2000 dilution (Cell Signaling Technology, 2146S); anti-GFP polyclonal antibody was used at 1:250 dilution (GeneTex, GTX113617); goat anti-mouse immunoglobulin G (IgG) secondary antibody, Alexa Fluor 546 (Thermo Fisher Scientific, A-11003); anti-goat anti-rabbit IgG secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-11008); anti-NXF1 monoclonal antibody was used at 1:1000 dilution (Abcam); anti-NP antibodies were used at 1:1000 dilution (GeneTex); rabbit polyclonal anti–SARS-CoV NP was used at 1:5000 dilution (produced at Mount Sinai); and rabbit anti-Nsp1 antibody was generated by Cocalico Biologicals using recombinant Nsp1 protein.

Techniques: Ubiquitin Proteomics, RNA Binding Assay, Binding Assay, Transfection, Fractionation, Immunoprecipitation, Western Blot, Expressing, Infection, In Vitro, Purification, Recombinant, Labeling

Journal: Science Advances

Article Title: Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression

doi: 10.1126/sciadv.abe7386

Figure Lengend Snippet: ( A ) 293T cells were transfected with plasmids encoding GFP, GFP-Nsp1, or GFP-Nsp1 and Flag-NXF1 and then analyzed by RNA-FISH to detect poly(A) RNA and immunofluorescence to detect GFP, GFP-Nsp1, and Flag-NXF1. White arrowheads show examples for each condition. Scale bars, 10 μm. ( B ) Fluorescence intensity of total poly(A) RNA in individual cells is shown in the depicted conditions. ( C ) Fluorescence intensity of poly(A) in the nucleus and cytoplasm was determined, and the ratios of the nuclear-to-cytoplasmic signals for individual cells are shown for the indicated experimental condition. n = 3; **** P < 0.0001, *** P < 0.001, and ** P < 0.01. ( D ) SK-N-SH cells were transfected with 3xFlag-Nsp1, and immunofluorescence microscopy was performed to detect Nsp1 and endogenous Nup358. n = 3. Scale bar, 5 μm. ( E ) Immunoprecipitation of Nsp1 followed by Western blot analysis shows Nsp1 interaction with certain nucleoporins, which is partially dependent on RNA.

Article Snippet: Serum containing anti-Tpr antibodies was used at 1:200 dilution, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (GeneTex, GTX627408) was used at 1:10,000 dilution; monoclonal mouse anti-NXF1 antibody was used at 1:1000 dilution (Sigma-Aldrich, T1076); polyclonal rabbit anti-NXF1 antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-915A); polyclonal rabbit anti-GANP antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-128A); polyclonal rabbit anti-THOC3 antibody was used at 1:1000 dilution (Bethyl Laboratories, A304-870A); polyclonal rabbit anti-nucleolin antibody was used at 1:1000 dilution (Novus Biologicals, NB600-241); polyclonal rabbit anti-UAP56 antibody was used at 1:1000 dilution (Sigma-Aldrich, SAB1307254); polyclonal rabbit anti-hnRNP K antibody was used at 1:2500 dilution (GeneTex, GTX101786); monoclonal rabbit anti-tubulin antibody was used at 1:2000 dilution (Cell Signaling Technology, 2146S); anti-GFP polyclonal antibody was used at 1:250 dilution (GeneTex, GTX113617); goat anti-mouse immunoglobulin G (IgG) secondary antibody, Alexa Fluor 546 (Thermo Fisher Scientific, A-11003); anti-goat anti-rabbit IgG secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-11008); anti-NXF1 monoclonal antibody was used at 1:1000 dilution (Abcam); anti-NP antibodies were used at 1:1000 dilution (GeneTex); rabbit polyclonal anti–SARS-CoV NP was used at 1:5000 dilution (produced at Mount Sinai); and rabbit anti-Nsp1 antibody was generated by Cocalico Biologicals using recombinant Nsp1 protein.

Techniques: Transfection, Immunofluorescence, Fluorescence, Microscopy, Immunoprecipitation, Western Blot

Journal: Science Advances

Article Title: Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression

doi: 10.1126/sciadv.abe7386

Figure Lengend Snippet: ( A ) Western blot analysis of proteins immunoprecipitated with NXF1 in the presence or absence of Nsp1 or RNA reveals that the interaction of NXF1 with key mRNA export adaptors and certain Nups is inhibited by Nsp1. ( B ) 293T cells expressing the SARS-CoV-2 receptor ACE2 protein were infected with SARS-CoV-2 at an MOI of 1 for 24 hours. NXF1 was specifically immunoprecipitated from the cell lysates and followed by Western blot analysis to detect nucleoporins and mRNA export factors. Association of NXF1 with constituents of the mRNA export machinery is inhibited during SARS-CoV-2 infection.

Article Snippet: Serum containing anti-Tpr antibodies was used at 1:200 dilution, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (GeneTex, GTX627408) was used at 1:10,000 dilution; monoclonal mouse anti-NXF1 antibody was used at 1:1000 dilution (Sigma-Aldrich, T1076); polyclonal rabbit anti-NXF1 antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-915A); polyclonal rabbit anti-GANP antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-128A); polyclonal rabbit anti-THOC3 antibody was used at 1:1000 dilution (Bethyl Laboratories, A304-870A); polyclonal rabbit anti-nucleolin antibody was used at 1:1000 dilution (Novus Biologicals, NB600-241); polyclonal rabbit anti-UAP56 antibody was used at 1:1000 dilution (Sigma-Aldrich, SAB1307254); polyclonal rabbit anti-hnRNP K antibody was used at 1:2500 dilution (GeneTex, GTX101786); monoclonal rabbit anti-tubulin antibody was used at 1:2000 dilution (Cell Signaling Technology, 2146S); anti-GFP polyclonal antibody was used at 1:250 dilution (GeneTex, GTX113617); goat anti-mouse immunoglobulin G (IgG) secondary antibody, Alexa Fluor 546 (Thermo Fisher Scientific, A-11003); anti-goat anti-rabbit IgG secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-11008); anti-NXF1 monoclonal antibody was used at 1:1000 dilution (Abcam); anti-NP antibodies were used at 1:1000 dilution (GeneTex); rabbit polyclonal anti–SARS-CoV NP was used at 1:5000 dilution (produced at Mount Sinai); and rabbit anti-Nsp1 antibody was generated by Cocalico Biologicals using recombinant Nsp1 protein.

Techniques: Western Blot, Immunoprecipitation, Expressing, Infection

Journal: Science Advances

Article Title: Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression

doi: 10.1126/sciadv.abe7386

Figure Lengend Snippet: ( A ) Vero E6 cells were infected with SARS-CoV-2 at an MOI of 1 for 48 hours. Fixed cells were treated with digitonin (1 μg/ml) to permeabilize the plasma membrane and examined by immunofluorescence microscopy to detect NXF1 and the viral NP protein. Scale bar, 10 μm. ( B ) The regions of the nuclear periphery were manually selected from equatorial sections of the nuclei in the z-stack images as indicated on the right, and the signal intensities along the NE were quantified ( n = 22 for mock and n = 19 for SARS-CoV-2). Individual measurements (black dots) and the summarized box and whisker plots were overlaid (left). The difference in the NXF1 intensities at the NE between the mock and the SARS-CoV-2 infection samples was tested using Student’s t test. ( C ) The whole-cell extracts from Vero cells mock or SARS-CoV-2 infected were analyzed by Western blotting using anti-NXF1, SARS-CoV-2 NP, and GAPDH antibodies at 48 hours postinfection. The positions of molecular mass makers are indicated. ( D to G ) Vero E6 cells were transfected for 24 hours with plasmids encoding full-length Flag-tagged NXF1 or the indicated mutants of NXF1 and then infected with SARS-CoV-2 at an MOI of 1 for 24 hours. Cells were examined by immunofluorescence (IF) microscopy to detect viral NP protein and Flag-tagged NXF1. Scale bars, 10 μm. ( H ) Percentage of infected cells (NP + ) was scored for each condition. Bars represent averages of three separate experiments, and dots represent the average of each of the three independent experiments. NXF1 (1–200), n = 831 cells; NXF1 (201–619), n = 1146 cells; and NXF1 (full-length), n = 1267 cells. ** P < 0.01 and * P < 0.05.

Article Snippet: Serum containing anti-Tpr antibodies was used at 1:200 dilution, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (GeneTex, GTX627408) was used at 1:10,000 dilution; monoclonal mouse anti-NXF1 antibody was used at 1:1000 dilution (Sigma-Aldrich, T1076); polyclonal rabbit anti-NXF1 antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-915A); polyclonal rabbit anti-GANP antibody was used at 1:1000 dilution (Bethyl Laboratories, A303-128A); polyclonal rabbit anti-THOC3 antibody was used at 1:1000 dilution (Bethyl Laboratories, A304-870A); polyclonal rabbit anti-nucleolin antibody was used at 1:1000 dilution (Novus Biologicals, NB600-241); polyclonal rabbit anti-UAP56 antibody was used at 1:1000 dilution (Sigma-Aldrich, SAB1307254); polyclonal rabbit anti-hnRNP K antibody was used at 1:2500 dilution (GeneTex, GTX101786); monoclonal rabbit anti-tubulin antibody was used at 1:2000 dilution (Cell Signaling Technology, 2146S); anti-GFP polyclonal antibody was used at 1:250 dilution (GeneTex, GTX113617); goat anti-mouse immunoglobulin G (IgG) secondary antibody, Alexa Fluor 546 (Thermo Fisher Scientific, A-11003); anti-goat anti-rabbit IgG secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-11008); anti-NXF1 monoclonal antibody was used at 1:1000 dilution (Abcam); anti-NP antibodies were used at 1:1000 dilution (GeneTex); rabbit polyclonal anti–SARS-CoV NP was used at 1:5000 dilution (produced at Mount Sinai); and rabbit anti-Nsp1 antibody was generated by Cocalico Biologicals using recombinant Nsp1 protein.

Techniques: Infection, Clinical Proteomics, Membrane, Immunofluorescence, Microscopy, Whisker Assay, Western Blot, Transfection