Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-β signalling by interacting with STAT1

Figures

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  Figure 1. Purified fusion protein and western blot analysis. A Lane 1, pET32a-UL47, non-induced; Lane 2, pET-32a-UL47 recombinant bacteria after induction in E. coli BL21 (DE3); Lane 3, pET-32a(+) vector; Lane 4, pET-32a-UL47 recombinant bacterial supernatant; Lane 5, pET-32a-UL47 recombinant bacterial sediment. B Purification of the recombinant protein. Lane 1, purified protein (approximately 55 kDa); C Expressed protein was recognized with rabbit anti-DEV antibody (1:800). Lane 1, recombinant protein (approximately 55 kDa); Lane 2, pET-32a (+) vector; M. Precision Plus Protein™ Dual Color Standards.

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  Figure 2. The DEV UL47 gene is a late gene. A Total RNA isolated from mock-infected and DEV-infected cells at various times was analysed using RT-qPCR and normalized to β-actin, and plotted the fold change over mock. B The expression analysis of the UL47 gene product in DEV-infected DEFs at different time points. The samples derived from the same experiment and gels were processed in parallel. C Schematic diagram showed the IE gene (UL54), E gene (UL13), and L gene (US2). D GCV group: DEV-infected cells treated with GCV; CHX group: DEV-infected cells treated with CHX. (+) group: DEV-infected cells without drugs. M, DL2000 Marker.

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  Figure 3. DEV pUL47 is a component of the virion. The purified DEV virions were identified using rabbit anti-UL47 antibody serum. As a control, no band was present in the DEF cell group.

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  Figure 4. pUL47 mainly localizes in the nucleus of DEV-infected cells. Representative DEV pUL47 localization (green in all images). The mock group included untreated DEF cells using rabbit anti-UL47 polyclonal antibody and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (Invitrogen, 1:1000) (magnification: 200×; scale bar: 10 μm).

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  Figure 5. The residues 40–50 and 768–777 of DEV pUL47 could direct heterologous proteins to the nucleus. All transfected samples were collected after 24 h posttransfection. A DEF cells were transfected with pEGFP-C2-SV40, pEGFP-C2-40-50, pEGFP-C2-768-777 and pEGFP-C2, respectively. B The expression of proteins was confirmed by Western blotting using mouse anti-GFP monoclonal antibody and goat anti-mouse IgG HRP-conjugated antibody (1:5000) (magnification: 200×; scale bar: 10 μm). C DEF cells were transfected with pEGFP-C2-SV40-β-gal, pEGFP-C2-40-50-β-gal, pEGFP-C2-768-777-β-gal and pEGFP-C2-β-gal, respectively. D The expression of proteins was confirmed by western blotting using mouse anti-GFP monoclonal antibody.

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  Figure 6. The residues 40 to 50 and 768 to 777 of DEV pUL47 are necessary for nuclear localization. A Distribution of the UL47 gene in DEF cells transfected with pcDNA3.1-Flag-UL47Δ40–50-768-777 (magnification: 200×; scale bar: 10 μm). B The expression of proteins was confirmed by western blotting using rabbit anti-UL47 polyclonal antibody.

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  Figure 7. DEV UL47 inhibits IFN-β signalling induced by poly(I:C). All transfected samples were collected at 36 h posttransfection. A DEV UL47 inhibited the IFN-β-luc luciferase activation induced by poly(I:C) in DEF cells. DEF cells were co-transfected with IFN-β-luc luciferase reporter plasmid, pRL-TK, pcaggs-UL47-Flag or an empty vector, and then stimulated by 50 µg/mL at 12 h posttransfection. The cells were harvested and detected by dual-luciferase assay at 24 h posttransfection. Protein expression was confirmed by western blotting using mouse anti-Flag MAb. All experiments were performed in triplicate (n = 3). The data was analyzed by one-way ANOVA. ***p < 0.001

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  Figure 8. pUL47 interacted with STAT1 in the cytoplasm and inhibited ISG expression induced by STAT1. A HEK293T cells were coexpressed pcaggs-STAT1-myc with pcaggs-UL47-Flag, pcaggs-STAT1-myc with an empty vector and pcaggs-UL47-Flag with an empty vector (magnification: 200×; scale bar: 10 μm). B BHK21 cells contained pcDNA3.1-C-UL47-LgBiT coexpressed with pcDNA3.1-N-STAT1-SmBiT, FKBP-SmBiT coexpressed with FRB-LgBiT as positive controls, Halotag-SmBiT coexpressed with pcDNA3.1-C-UL47-LgBiT, pcDNA3.1-C-UL47-LgBiT coexpressed with FKBP-SmBiT and pcDNA3.1-N-STAT1-SmBiT coexpressed with FRB-LgBiT as negative controls. The luciferase signal was detected via NanoBiT after 20-h posttransfection. C DEV UL47 inhibited the mRNA levels of Mx and OASL induced by STAT1. DEF cells were co-transfected with pcaggs-STAT1-myc and pcaggs-UL47-Flag or an empty vector. The cells were harvested and detected by RT-qPCR at 36 h posttransfection. Protein expression was confirmed by Western blotting. All experiments were performed in triplicate (n = 3). The data was analyzed by one-way ANOVA. *P < 0.05, **P < 0.01 and ****P < 0.0001.