Inhibition of RAN attenuates influenza a virus replication and nucleoprotein nuclear export

Abstract

Nuclear export of the viral ribonucleoprotein (vRNP) is a critical step in the influenza A virus (IAV) life cycle and may be an effective target for the development of anti-IAV drugs. The host factor ras-related nuclear protein (RAN) is known to participate in the life cycle of several viruses, but its role in influenza virus replication remains unknown. In the present study, we aimed to determine the function of RAN in influenza virus replication using different cell lines and subtype strains. We found that RAN is essential for the nuclear export of vRNP, as it enhances the binding affinity of XPO1 toward the viral nuclear export protein NS2. Depletion of RAN constrained the vRNP complex in the nucleus and attenuated the replication of various subtypes of influenza virus. Using in silico compound screening, we identified that bepotastine could dissociate the RAN-XPO1-vRNP trimeric complex and exhibit potent antiviral activity against influenza virus both in vitro and in vivo. This study demonstrates the important role of RAN in IAV replication and suggests its potential use as an antiviral target.

Keywords: Influenza A virus; RAN; bepotastine; nuclear export; viral ribonucleoprotein.

Figure 1.

Knockdown of RAN by siRNA inhibits IAV replication in avian cells. (A) The knockdown efficiency of the siRNAs in DF-1 cells was validated by western blotting (left). Viability of siRAN-1-transfected DF-1 cells were measured using the cell counting kit 8 (CCK-8) (right). (B–I) DF-1 or CEF cells were transfected with siRAN or control siRNAs for 24 h, followed by infection with the H5N6-JX, H5N1-DW or H7N9-GX viruses at an MOI of 0.01. Cell supernatants were collected at different time points post-infection, as indicated, and assayed for virus titres by TCID₅₀ (panels B, D, F and H). RAN and viral proteins were probed using western blotting (panels C, E, G and I). (J and K) DF-1 cells were transfected with empty vector or Flag-RAN for 24 h, followed by the infection with H5N6-JX (MOI = 0.01). Cell supernatants and lysates were harvested for the TCID₅₀ and western blotting assays. (L) A variant of RAN with the synonymous mutation (Flag-RAN-M) was designed to avoid targeting by the adopted siRAN. (M and N) siRAN was co-transfected with the empty vector or Flag-RAN-M, followed by infection with the H5N6-JX virus (MOI = 0.01). The virus titres and viral proteins were assessed via the TCID₅₀ assay and western blotting, respectively. (O) DF-1 cells were infected with the H5N6-GFP virus for 24 h after transfection with siRNAs, and the GFP signal of the infected cells was determined using immunofluorescence microscopy. Scale bar: 400 μm. Data were collected from three independent experiments and presented as mean ± SD. Statistical significance was determined using a two-way ANOVA. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.

RAN is essential for nuclear export of vRNP. (A and B) siRNA-transfected DF-1 cells were infected with H5N6-JX at an MOI of 10 in the presence of CHX (100 μg/mL). Cells were fixed and permeabilized at 2 h or 6 h post-infection and then immunostained for NP. Baf-A1 (100 nM) and leptomycin (10 ng/mL) were used as nuclear import and export inhibition controls, respectively. The images were acquired under confocal microscopy. Scale bar: 20 μm. (C) siRNA-transfected DF-1 cells were infected with H5N6-JX at an MOI of 10. M1 protein of virus particles attached to or entered into the cells was evaluated by western blotting, in which 8 h sialidase pretreatment before infection was set as a control. (D) DF-1 cells were transfected with siRAN or control siRNAs for 24 h, followed by infection with the H5N6-JX at an MOI of 10 for the specific time points, the infected cells were fixed and stained successively with an anti-NP antibody and DAPI. (E) DF-1 cells transfected with siRNAs were infected with H5N6-JX at an MOI of 10. Cells were fixed at 4, 6 and 8 h post-infection, and the location of M vRNAs was detected using fluorescence in situ hybridization. (F and G) The cells at 4, 6 and 8 h post-infection in D and E with different NP protein or M vRNA distribution were counted for quantitative analysis. N, predominantly nuclear; C+N, nuclear and cytoplasmic; C, predominantly cytoplasmic. The results shown are calculated from one hundred cells viewed under a confocal microscope. Scale bar: 20 μm. The results are representative of at least three independent experiments.

Figure 3.

RAN interacts with XPO1 and enhances the binding of XPO1 with viral NS2. (A and B) DF-1 cells were co-transfected with Flag-RAN and HA-XPO1. The co-IP experiment was performed using an anti-Flag or an anti-HA antibody, followed by western blotting analysis. (C) The localization of RAN (green) and XPO1 (red) was assessed via an indirect immunofluorescence assay using confocal microscopy. Scale bar: 20 μm. (D and E) Endogenous interaction between RAN and XPO1 was analyzed. DF-1 cells from two 10-cm dishes were lysed with 500 μL IP lysis buffer, and immunoprecipitation was performed using anti-RAN, anti-XPO1 or control IgG antibodies. The pull-down products were analyzed using western blotting. (F and G) DF-1 cells were infected with H5N6-JX at an MOI of 5 in the presence or absence of siRAN. At 6 h post-infection, the co-IP experiment was performed using anti-XPO1, anti-NS2 or control IgG antibodies, followed by western blotting. (H and I) The siRNA-transfected DF-1 cells were co-transfected with EGFP-NS2, along with the HA-XPO1 or HA-empty vector; then, the co-IP experiment was performed using anti-HA, anti-GFP or control IgG antibodies, followed by western blotting. (J) XPO1-NS2 or XPO1-NS2-RAN interaction energy, RMSD (K) and BSA (L) are plotted over the course of the 600 ps extended MD simulation. The results are representative of at least three independent experiments.

Figure 4.

RAN knockdown restrains the replication of influenza virus both in human cells and mice. (A–F) A549 cells were transfected with siRAN or siNC and then infected with various subtypes of influenza virus at an MOI of 10 or 0.01. At 8 h post infection, the NP and nuclei were stained with an anti-NP antibody and DAPI, respectively. Images were obtained using confocal microscopy. Scale bar: 20 μm. At 24 h post-infection, viral titres in the supernatant and NP protein in the cells were determined using the TCID₅₀ and western blotting. (G) Two siRNAs targeting mouse RAN were delivered to mice, and the RAN protein levels in the mouse lungs were detected via western blotting. (H) The integrated OD (IOD) of RAN and Actin in G was measured using the Image-Pro Plus software, and the relative IOD was generated using IOD RAN/IOD Actin. (I) Schematic of the in vivo experiment for evaluating the antiviral effect of RAN knockdown on PR8 (n = 7). (J) Weight loss and survival (K) were evaluated in the PR8- and siRNA-treated mice. Data were collected from three independent experiments and are presented as mean ± SD. Statistical significance was determined using a two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5.

Bepotastine as an effective inhibitor of IAV replication. Schematic representation of the screening strategy to obtain RAN inhibitor candidates (created with BioRender.com with RAN structure from PDB, ID: 4HAT). (B and C) 3D models (B) and the corresponding 2D interaction diagram (C) generated through ligand-target modelling studies. (D and E) Quantitative luciferase activity and cytotoxicity testing (CCK-8 assay) of IAV replication levels in A549 (D) and MDCK cells (E) infected with PR8-Gluc viruses (MOI = 0.01) for 24 h at the indicated concentrations. (F and G) A549 and MDCK cells infected with PR8 were incubated with bepotastine (20 μM), oseltamivir (20 μM), or culture medium (negative control) for 12, 24 or 36 h. The virus titres and viral nucleoproteins were assayed using TCID₅₀ and western blotting. (H and I) A549 and MDCK cells infected with PR8 were treated with various concentrations of bepotastine (5, 10 and 20 μM), oseltamivir (20 μM), or ordinary medium (negative control) for 24 h. The virus titres and viral nucleoproteins were assayed using TCID₅₀ and western blotting. (J and K) The human primary alveolar epithelial cells (J) and NK cells (K) treated with bepotastine (20 μM) were infected with PR8 at an MOI of 0.1 for 24 h. The virus titres and viral nucleoproteins were assayed using TCID₅₀ and western blotting. Data were collected from three independent experiments and are presented as mean ± SD. Statistical significance was determined using a two-tailed Student’s t-test (H, I, J and K) or a two-way ANOVA (F and G). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6.

Bepotastine inhibits vRNP nuclear export via interaction with cellular RAN and exhibits antiviral effects in human primary cells. (A and B) A549 cells treated with bepotastine (30 μM) and leptomycin (10 ng/mL) were infected with the indicated viruses at an MOI of 10. At 8 hpi, the distribution of NP was analyzed by IFA under confocal microscopy (A), Scale bar: 20 μm. The cells with different NP distribution were counted for quantitative analysis (n = 100) (B). (C) The NP in the cytoplasmic and nuclear fractions was subjected to western blotting analysis. (D and E) Bepotastine induced NP nucleus retention was assessed in the human primary alveolar epithelial cells by IFA. Scale bar: 20 μm. (F) Streptavidin pulldown assay of biotin or biotin-bepotastine (10 mM) (a) and purified GST-RAN (5 μg/mL). (b) Purified GST and GST-RAN proteins were detected by Coomassie blue staining, and the bound proteins were analyzed by western blotting. (G) Endogenous expressed RAN was pulled down by biotin-bepotastine. (H and I) The bepotastine (10 and 20 μM) treated A549 cells were co-transfected with HA-huXPO1 and Flag-huRAN for 24 h, then the co-IP experiment was performed using anti-HA or anti-Flag antibodies, followed by western blotting assays. (J) GST pulldown assays by purified wildtype or mutated GST-huRAN proteins together with the lysates of HA-XPO1 expressing HEK293 T cells in the absence or presence of bepotastine (10 mM). The results are representative of at least three independent experiments.

Figure 7.

Bepotastine restricts PR8 replication in mice. (A) Schematic diagram of the in vivo experiments evaluating the antiviral effect of bepotastine against PR8. (B–G) Female BALB/c mice were inoculated with 50 μL of 10 LD₅₀ PR8 and treated with bepotastine (2, 10 and 50 mg/kg), oseltamivir (10 mg/kg) or 1% DMSO. In the next 2 weeks, the body weights (B) and mouse survival (C) were recorded daily. On 5 dpi, the right lungs were fixed and subjected to detect NP by immunohistochemistry (D) and the viral titres of the left lungs were measured by TCID₅₀ determination (E). The right lung injury of the mice was assessed by H&E staining at 5 dpi (F) and the blinded sections were scored for levels of pathological severity (G). The following scoring system was used: 0, no pathological change; 1, affected area (≤ 10%); 2, affected area (< 50%, > 10%); 3, affected area (≥ 50%). Data were collected from three independent experiments and presented as the mean ± SD. Statistical significance was determined using a two-tailed Student’s t-test. *p < 0.05; **p < 0.01.

Figure 8.

Model for RAN-regulated vRNP nuclear export during influenza virus infection. Late in infection, the mature vRNPs are transported from the nucleus to the cytoplasm for the assembly of progeny virus. Naturally, RAN promotes the nuclear export of vRNP by enhancing the interaction between XPO1 and NS2. When RAN is absent or is inhibited by a small molecule like bepotastine, the conformation of XPO1 is intolerable for NS2-vRNP binding, which largely blocks the vRNP nuclear export.