Importin-7-dependent nuclear translocation of the Flavivirus core protein is required for infectious virus production

Abstract

Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-β family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses.

Fig 1. In vitro biochemical analysis of the nuclear localization of flavivirus core proteins.

(A) The plasmid coding non-tagged JEV core or DENV core was transfected in Huh7 cells. After two days, transfectants were fixed and stained with the anti-JEV core or anti-DENV core antibody. The scale bar indicates 20 μm. The right graph shows the quantification of the ratio of nuclear translocation. (B) Recombinant AcGFP-core of each virus was expressed in the baculovirus expression system and visualized by SDS-PAGE and CBB stain. (C) Experimental design for the analysis of the nuclear localization of core proteins. In the injection (IJ) assay, recombinant core proteins and immunoglobulin (IgG) were injected into Huh7 cells. After 30 min, the localization of recombinant core proteins was observed through confocal laser microscopy. In the permeabilization (PM) assay, Huh7 cells were treated with digitonin, and the permeabilized cells were then incubated with recombinant core proteins. After 30 min, the localization of recombinant core proteins was observed through confocal laser microscopy. (D) Localization of core proteins of flaviviruses by the IJ assay. Recombinant AcGFP-core protein and IgG labeled with Alexa Fluor 555 were injected into Huh7 cells. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. (E) PM assay using GST and GFP fused with the NLS of SV40 large T antigen (SV40 NLS) or AcGFP-JEV core WT or AcGFP-JEV core GP/AA. Rabbit reticulocyte lysate was employed as a cytosol source including import factors. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. All data are representative of three independent experiments. Significance (*p < 0.05; **p < 0.01; n. s., not significant) was determined using Student’s t-test (n = 3). Created with Biorender.com.

Fig 2. Nuclear pore complex (NPC)-mediated nuclear localization of core proteins is importin-α-independent and importin-β-dependent.

(A) Scheme of the experiment used to assess the involvement of importin-α (Impα), importin-β (Impβ), and NPC. (B) The injection (IJ) assay was used to assess the involvement of importin-α, importin-β, and NPC in core protein nuclear localization. Bimax, RanGTP, and WGA were used as inhibitors of importin-α, importin-β, and nuclear pore complexes, respectively. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. (C) A GST pull-down assay was performed using GST-fused importin-β1 mixed with flag-tagged core protein or importin-α1, respectively. Input and immunoprecipitant samples were subjected to SDS-PAGE and detected by western blotting using anti-GST and anti-FLAG antibodies. All data are representative of three independent experiments. Significance (*p < 0.05; **p < 0.01; n. s., not significant) was determined using Student’s t-test (n = 3). Created with Biorender.com.

Fig 3. Importin-7 (IPO7) specifically interacts with the Japanese encephalitis virus (JEV) core protein.

(A) AcGFP-FOS or AcGFP-JEV core-FOS was transfected into 293T cells and precipitated using Strep-Tactin Sepharose beads. The pull-down samples were analyzed through mass spectrometry (MS). (B) List of importin-β family proteins identified by MS analysis. The numbers of unique peptides identified by MS are indicated. (C) AcGFP-FOS or AcGFP-JEV core-FOS was transfected into 293T cells and precipitated using Strep-Tactin Sepharose beads. The precipitated samples were subjected to SDS-PAGE and detected by western blotting using anti-IPO7 and anti-flag antibodies. The data are representative of three independent experiments.

Fig 4. Importin-7 (IPO7) can transport flavivirus core proteins into the nucleus.

(A) GST or GST-IPO7 was incubated with AcGFP-RPS7 in the presence or absence of 100 pmol or 770 pmol RanGTP Q69L. The pulled-down proteins were subjected to SDS-PAGE and detected by western blotting using anti-IPO7 and anti-GFP antibodies. The numbers indicate amounts of each protein calculated using the Image J software. (B) Either GST or GST-IPO7 was incubated with AcGFP-JEV core with or without 100 pmol or 770 pmol RanGTP Q69L. The pulled-down proteins were subjected to SDS-PAGE and detected by western blotting using anti-IPO7 and anti-GFP antibodies. The numbers indicate amounts of each protein calculated using the Image J software. (C) The permeabilization (PM) assay was performed using recombinant IPO7 protein. Digitonin-permeabilized Huh7 cells were incubated with GST-GFP-RPL23A or AcGFP-JEV core proteins with or without IPO7. Localization of core protein was observed through confocal laser microscopy. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. (D) IPO7 antibody or control IgG was injected with recombinant Japanese encephalitis virus (JEV) core protein and IgG conjugated with Alexa Fluor 594. Core protein localization was observed through confocal laser microscopy. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. All data are representative of three independent experiments. Significance (*p < 0.05; **p < 0.01; n. s., not significant) was determined using Student’s t-test (n = 3).

Fig 5. IPO7 deficiency in Huh7 cells impairs the nuclear translocation of core proteins of Flaviviridae.

(A) GST and GFP fused the SV40 large T antigen NLS, or AcGFP fused core proteins of Flaviviridae were microinjected into the cytoplasm of WT or KO cells with red fluorescence protein-conjugated antibody as a cytoplasmic injection marker. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. (B) Plasmids encoding AcGFP fused with core proteins of Flaviviridae were transfected into wild-type (WT) or IPO7-KO Huh7 cells. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. (C, D) Plasmids encoding non-tagged JEV or DENV core proteins were transfected into WT or IPO7-KO Huh7 cells. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. (E, F) The WT or IPO7-KO Huh7 cells were infected with JEV and DENV. Subcellular localization of core protein was observed using indicated antibodies. The scale bar indicates 20 μm. The right graph is the quantification of the ratio of nuclear translocation. All data are representative of three independent experiments. Significance (*p < 0.05; **p < 0.01; n. s., not significant) was determined using Student’s t-test (n = 3).

Fig 6. IPO7 affects infectious viral release in the viral life cycle.

(A, B) WT or IPO7-KO Huh7 cells were infected with JEV at a multiplicity of infection (MOI) of 5. The viral titers in supernatants (A) and intracellular viral RNA (B) were quantified at each time point. (C) WT or IPO7-KO Huh7 cells infected with JEV at an MOI of 5 were collected three days post-infection. Intracellular viral titers were determined after each of repeated freeze–thaw cycles. (D) WT or IPO7-KO Huh7 cells were infected with JEV. Viral titers were determined for each virus. WT Huh7 cells were infected with the virus at a multiplicity of infection (MOI) of 5. After 2 days, the supernatant was collected, and the viral titer was determined. (E) WT or IPO7-KO Huh7 cells were infected with JEV at an MOI of 5. The supernatants were collected at 2 days post infection. Each viral supernatant was infected into Huh7 cells at MOI of 5. After 2 days, each supernatant was collected and was determined viral titer. (F) WT JEV or mutant JEV harboring GP/AA core was infected at an MOI of 1 into WT or IPO7-KO Huh7 cells. The supernatants were collected at 2 days post infection and determined the viral titers. (G, H) WT or IPO7-KO Huh7 cells were infected with ZIKV and DENV at a multiplicity of infection (MOI) of 5. The viral titers in supernatants (G) and intracellular viral RNA (H) at three days post-infection were quantified. All data are representative of three independent experiments. Significance (*p < 0.05; **p < 0.01; n.s., not significant) was determined using Student’s t-test (n = 3).

Fig 7. Disruption of the nuclear localization of the core proteins of Flaviviridae impairs efficient infectious virus release.

(A) Schematic procedure of the single-round infectious particle (SRIP) generation experiments. (B) Three different plasmids containing the core, prME, and replicon were transfected into 293T cells to produce SRIPs. SRIPs produced by wild-type (WT) or GP/AA core protein were used to infect Vero cells. Nluc reporter activity was quantified using the Nano-Glo Luciferase Assay System. (C) SRIPs harboring WT or GP/AA JEV core were generated from WT or IPO7-KO Huh7. These SRIPs were infected Vero cells. Nluc reporter activity was quantified using the Nano-Glo Luciferase Assay System. (D) The core, prME, and replicon plasmids were transfected into WT or IPO7-KO Huh7. Intracellular SRIPs were collected after each repeat of freeze–thaw cycles. (E) In vitro-transcribed JEV replicon RNA was electroporated into WT and IPO7-KO Huh7 cells. Viral RNA 4 and 48 h post-transfection was calculated based on Nluc activity at 4 h post-electroporation. (F) SRIPs of Zika virus (ZIKV), dengue virus (DENV), or yellow fever virus (YFV) were generated from WT or IPO7-KO Huh7 cells. The reporter activity of the SRIPs was determined after infection into Vero cells. All data are presented as the mean ± SD of three independent experiments. Significance (**p < 0.01; n.s., not significant) was determined using Student’s t-test (n = 3; B-D and F, n = 4; E). Created with Biorender.com.