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. 2022 Aug 15;13:4782. doi: 10.1038/s41467-022-32489-5

Fig. 4. Interactions with Rae1-Nup98 are important for ORF6-mediated disruption of nucleocytoplasmic transport.

Fig. 4

a HEK-293T cells were co-transfected with the indicated plasmids, and 48 h later, Co-IP analysis was performed to examine the interaction between Rae1-Nup98 and wild-type (WT) or mutant viral proteins. Results shown are representative of three independent experiments. b HEK-293T cells were transfected with pEGFP-C1 and individual plasmid expressing WT or mutant ORF6, ORF10, or M. GFP expression were analyzed by fluorescence microscopy. All fluorescence images at 40 times magnification (40×). Results shown are representative of three independent experiments. c Quantification of the fluorescence intensity in b with Image J. Data are representative of three independent experiments and shown as the mean ± SD. (*p < 0.05, ***p < 0.001, ****p < 0.0001; two tailed Student’s t-test, n = 3). d The cytoplasm and nuclear RNAs were extracted from transfected cells in b, and the levels of GFP transcripts were quantified by RT-PCR. The Nu/Cyto ratio is plotted as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two tailed Student’s t-test, n = 3). e, f The effects of ORF6 mutants on ISRE-promoter activation. HEK-293T cells were transfected with individual plasmid expressing WT or mutant ORF6, ISRE-luc and TK-Renilla reporter plasmids, and 24 h later, treated with IFN-α and IFN-β for 16 h. Dual luciferase reporter assay was conducted. Data are representative of three independent experiments and shown as the mean ± SD. (**p < 0.01, ***p < 0.001, ****p < 0.0001; two tailed Student’s t-test, n = 3). g The effects of ORF6 mutants on STAT1 nuclear translocation. HEK-293T cells were transfected with individual plasmid expressing WT or mutant ORF6, and 24 h later, the cells were treated with IFN-β (500 IU/ml) for 1 h. The nuclear and cytoplasmic fractions were separated for Western blotting analysis. Results shown are representative of three independent experiments. h Quantification of the expression levels of STAT1 in nuclear and cytoplasmic fractions in g by Image J. i Subcellular localization of pSTAT1. HEK293 cells were transfected with individual plasmid expressing WT, mutant Flag-tagged ORF6, or empty vector (EV). 24 h post-transfection, cells were treated with IFN-β (500 IU/ml) for 1 h. ORF6 and endogenous pSTAT1 were analyzed by confocal microscopy. Scale bars, 10 μm. Results shown are representative of three independent experiments. Source data are provided as a Source data file.