Fig 2: Nsp1 prevents proper interaction of NXF1 with the mRNA export machinery.(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.

Fig 3: TPR is required for localization of TREX-2 complex at the NPC.a–c Both TPR and GANP are required for PCID2 and ENY2 localization at the NE. Localization of mCherry-PCID2 and mCherry-ENY2 in cells after acute loss of TPR (a), NUP153 (b), or GANP (c). (see also Supplementary Fig. 7b, c). Note the changes in NPC localization of PCID2 and ENY2 (yellow arrowheads). Scale bar is 7 µm. All BSK-NUPs and NXF1 AID-tagged cell lines were treated with auxin for 2 h; GANP AID-tagged cell line was treated with auxin for 3 h. Data are representative of three independent experiments. d A schematic representation of the TREX-2–NPC interaction.

Fig 4: NXF1 localization at the NPC is reduced upon SARS-CoV-2 infection and increased NXF1 level prevents infection.(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.

Fig 5: NXF1 expression reverts Nsp1-mediated mRNA export block and reduction in mRNA levels.(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.