Fig. 6: ORF6 modestly disrupts the importin α/β1 pathway.

a Subcellular localization of mCherry-NLS (red) in HeLa cells transfected with AcGFP or AcGFP-ORF6 (green). DAPI was used to stain the DNA (blue). Scale bars: 30 μm. b The graph represents the relative fluorescence values of the nucleus compared to those of the whole cells in a. Signal intensities of total 50 nuclei from two independent experiments were measured. ***P < 0.001, two-tailed Student’s t-test. error bars represent SD. c Importin α1 (Impα1) was incubated with GST-GFP, GST-GFP-ORF6, or GST-NLS-GFP immobilized on GST-beads for 1 h, and then a pulled-down protein was collected (PD). The importin α1 proteins were detected using the anti-importin α1 antibody (WB). The bottom panel represents the proteins bound to the beads and stained with CBB. The right lane was loaded importin α1 as an input protein (1/30,000 dilution of the reaction). Values are kDa. d An in vitro semi-intact nuclear transport assay was performed to measure the nuclear import of GST-NLS-mRFP in the presence of AcGFP-ORF6. Digitonin-permeabilized HeLa cells were incubated with GST-NLS-mRFP, importin α1, importin β1, RanGDP, p10/NTF2, GTP, and ATP regeneration system. The reaction mixture was added 5×, 10×, or 20× concentration of AcGFP-ORF6 compared to that of the NLS-substrate. After incubation for 30 min, the mRFP signals were detected using a fluorescence microscope. DAPI was used to stain the DNA. Scale bars: 30 μm. e The graph represents the nuclear fluorescence values of GST-NLS-mRFP in d. Signal intensities of total 100 nuclei were measured and analyzed using a one-way ANOVA (***P < 0.001). error bars represent SD. f, g Immunofluorescence of either HIF-1α f or NF-κB p65 g in HeLa cells transfected with AcGFP or AcGFP-ORF6 following CoCl2 treatment or TNF-α stimulation, respectively. Anti-GFP, anti-HIF-1α or anti-p65 antibodies were used for detection of AcGFP (green), HIF-1α (red), or p65 (red), respectively. DAPI was used to stain the DNA (blue). Scale bars: 30 μm. h, i The graphs represent the relative fluorescence values of HIF-1α h or NF-κB p65 (i) in the nucleus compared to those of the entire cells based on in f or g, respectively. Signal intensities of total 50 nuclei from two independent experiments were measured. ***P < 0.001, two-tailed Student’s t-test. error bars represent SD. j Immunofluorescence of NF-κB p65 in Vero-TMPRSS2 cells infected with SARS-CoV-2 following TNF-α stimulation. Endogenous p65 (green) and viral ORF6 (red) were detected using the specific antibodies. k The graph represents the relative fluorescence values of p65 in the nucleus compared to whole cells in j. Signal intensities of a total of 80 nuclei from two independent experiments were measured. Two-tailed Student’s t-test. n.s.: not significant. error bars represent SD. Scale bars: 30 μm.