Dual function of the nuclear export signal of the Borna disease virus nucleoprotein in nuclear export activity and binding to viral phosphoprotein

Figures

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  Fig. 1. Minigenome assay of BoDV-N NES mutants. Schematic diagram of BoDV-N (a) and BoDV minigenome (b). c. Minigenome assay using BoDV-N NES mutants. 293 LTV cells were transfected with pC-BoDV-MG-Gluc, pC-BoDV-N, pC-BoDV-L, and pCXN2-P. At 48 h post-transfection, luciferase activity was measured. The experiments were performed independently three times. The data were analyzed with Student’s t-test. Bars show mean ± standard deviation (SD) (*; p value <0.05)

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  Fig. 2. Subcellular localization of BoDV-N NES mutants. a. 293 T cells were transfected with pC-BoDV-MG-Gluc, pC-BoDV-N, pC-BoDV-L, and pCXN2-P. b. 293 T cells were transfected with pC-BoDV-MG-Gluc, pC-BoDV-L, pCXN2-P and 87 ng of pC-BoDV-N (WT) or 2.1 μg of pC-BoDV-N-NES-A (ALL). a and b. At 48 h post-transfection, the cells were subjected to biochemical fractionation. BoDV-N and the fraction marker proteins (snRNP70 for the nucleus and β-tubulin or GAPDH for the cytoplasm) were detected by western blotting using anti-BoDV-N HN132 monoclonal antibody and anti-snRNP70 polyclonal, anti-β-tubulin, or anti-GAPDH monoclonal antibodies, respectively

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  Fig. 3. Interaction of BoDV-N NES mutants with BoDV-P. a. 293 T cells were transfected with pC-FLAG-BoDV-N, pC-BoDV-L, and pCNX2-HA-BoDV-P. At 48 h post-transfection, the cells were subjected to an IP assay using anti-FLAG M2 monoclonal antibody. The IP product was analyzed by western blotting using anti-FLAG M2 polyclonal and anti-HA monoclonal antibodies. b. Nuclear localization of BoDV-Np40 containing L131A mutation. OL cells were transfected with BoDV-Np40- or BoDV-Np40-L131-expression plasmids. At 48 h post-transfection, the cells were subjected to IFA using an anti-BoDV-N HN132 monoclonal antibody, and the percentages of cells expressing N only in the nucleus were calculated. A total of 150 cells were counted in 10 fields. The experiments were performed independently three times. The data were analyzed with Student’s t-test. Bars show mean ± standard deviation (SD) (*; p value <0.05)

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  Fig. 4. Characterization of rBoDV P/M-GFP-L131A. a. Expression levels of viral proteins in infected cells. Western blotting was performed with anti-BoDV-N HN132 monoclonal, anti-BoDV-P polyclonal, BoDV-X polyclonal, and anti-β-tubulin monoclonal (Sigma-Aldrich) antibodies. Expression levels of genomic RNA (b) and viral mRNA (c) in infected cells. Total RNA was extracted from infected cells and subjected to RT-qPCR. The experiments were performed independently three times. The data were analyzed with Student’s t-test. Bars show mean ± SD. d. Growth kinetics of rBoDV P/M-GFP-L131A. OL cells were infected with rBoDV P/M-GFP-L131A (L131A) or rBoDV P/M-GFP (WT) at a multiplicity of infection of 1. At days 3, 15, 20, 30, 40, and 50 post-infection, GFP-positive cells were counted using a Tali Image-Based Cytometer (Thermo Fisher Scientific). The experiments were performed independently three times. Bars show mean ± SD