GTPase domain of porcine Mx1 protein interacts with the N protein of porcine deltacoronavirus to inhibit viral replication

Figures

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  Fig. 1.. pMx1 is an ISG induced by type I interferon, and PDCoV infection upregulates pMx1 expression in IPEC-J2 cells and IPI-2I cells. IPEC-J2 cells (a–c) were treated with IFN-α or IFN-λ (1.85×10² U), and pMx1 expression levels at different time points, with or without stimulation, were analysed by RT-qPCR and Western blot. IPEC-J2 cells (d, e) and IPI-2I cells (f, g) were infected with PDCoV at an m.o.i. of 0.5, and cell lysates were collected at the indicated time points post-infection. Changes in pMx1 mRNA and protein levels were determined by RT-qPCR and Western blot. Data are presented as the mean±sd of three independent experiments. Statistical significance was analysed using two-way ANOVA followed by Bonferroni’s post-hoc test between IFN-treated groups and the mock control. *P<0.05; **P<0.01; ****P<0.0001; ns, not significant.

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  Fig. 2.. (a, b) IPEC-J2 and IPI-2I cells were seeded in 12-well plates and transfected with equal amounts of the pMx1 expression plasmid (pCAGGS-N-Flag-pMx1). At 24 h post-transfection, cells were infected with PDCoV. After 24 h of infection, cells were fixed with 4% paraformaldehyde, and PDCoV infection levels were evaluated by IFA and CPE observation. (c, d) Following pMx1 overexpression in IPEC-J2 and IPI-2I cells, cells were infected with PDCoV, and supernatants were collected at the indicated time points to determine viral titres (log-transformed values; mean±sd; n=3). Statistical significance was analysed using two-way ANOVA followed by Bonferroni’s post-hoc test between pMx1-overexpressing groups and the mock control. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant. (e–j) IPEC-J2 and IPI-2I cells were transfected with the pMx1 expression plasmid and subsequently infected with PDCoV. Cell lysates were collected at the indicated time points post-infection, and changes in PDCoV N mRNA and protein levels were analysed by RT-qPCR and Western blot. Data are presented as the mean±sd of three independent experiments. Statistical analysis was performed using a two-tailed…

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  Fig. 3.. (a, b) Three siRNAs targeting pMx1 were designed. The siRNA showing the highest knockdown efficiency was selected based on RT-qPCR analysis and further confirmed by Western blot. Data are presented as the mean±sd of three independent experiments. Statistical significance was analysed using one-way ANOVA with Dunnett’s post-hoc test. **P<0.01; ns, not significant. (c–h) IPI-2I and IPEC-J2 cells were transfected with siRNA-pMx1-3 or negative control (NC), followed by infection with PDCoV (m.o.i.=0.5). At 24 h post-infection, the mRNA and protein levels of PDCoV N were analysed by RT-qPCR and Western blot. (i, l) IPI-2I and IPEC-J2 cells were transfected with the pMx1 expression plasmid in 12-well plates. At 24 h post-transfection, cells were pre-chilled for 30 min and then incubated with PDCoV (m.o.i.=10) at 4 °C for 1 h to allow viral attachment. Cells were washed three times with pre-cooled PBS to remove unbound virus, and intracellular viral RNA levels during the attachment stage were determined by RT-qPCR. Data are presented as the mean±sd of three independent experiments. Statistical analysis was performed using a two-tailed unpaired Student’s t-test. *P<0.05; **P<0.01;…

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  Fig. 4.. The PDCoV N protein interacts with pMx1 and co-localizes in the perinuclear cytoplasm. (a–c) In HEK293T cells, His-PDCoV S was co-transfected with FLAG-pMx1, Myc-PDCoV N was co-transfected with FLAG-pMx1, and HA-PDCoV M was co-transfected with FLAG-pMx1; transfection of His-PDCoV S, HA-PDCoV M, Myc-PDCoV N, or FLAG-pMx1 alone was left as a control. At 36 h post-transfection, the cells were collected and lysed for co-immunoprecipitation (Co-IP) analysis with anti-FLAG (a, b) or anti-Myc (c) antibodies, respectively. (d) In HEK 293 T cells, FLAG-pMx1 and Myc-PDCoV N were co-transfected, while FLAG-pMx1 or Myc-PDCoV N alone were transfected as controls. Immunofluorescence staining was then performed, and cells were examined by confocal microscopy to determine the localization of PDCoV N protein (green), pMx1 (red), and the nucleus (blue).

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  Fig. 5.. The GTPase domain of pMx1 is required for its interaction with PDCoV N. (a) Schematic diagram of pMx1 protein domains and the positions of the truncation mutants used in this study. (b–d) HEK293T cells were co-transfected with Myc-PDCoV N and FLAG-tagged pMx1 truncation constructs (FLAG-GD, FLAG-C-pMx1, or FLAG-GMD). Cells transfected with FLAG-GD, FLAG-C-pMx1, FLAG-GMD or Myc-PDCoV N alone served as controls. At 36 h post-transfection, cells were harvested and lysed for Co-IP assays using anti-Myc antibodies, as indicated. (e) Subcellular localization of PDCoV N and the pMx1 truncation mutants in HEK293T cells was examined by confocal microscopy following co-transfection. (F–I) Equal amounts of full-length pMx1, its truncation mutants, or empty vector were transfected into IPEC-J2 and IPI-2I cells. At 24 h post-transfection, cells were infected with PDCoV (m.o.i.=0.5). At 24 h post-infection, the mRNA and protein levels of PDCoV N in cell lysates were analysed by RT-qPCR and Western blot. Data are presented as the mean±sd of three independent experiments. Statistical significance was analysed using one-way ANOVA with Dunnett’s post-hoc test. **P<0.01; ***P<0.001; ****P<0.0001;…

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  Fig. 6.. PDCoV N interacts with nsp12 and co-localizes with it in the perinuclear region, and pMx1 attenuates their interaction. (a) HEK293T cells were co-transfected with HA-nsp12 and Myc-PDCoV N expression plasmids. Cells transfected with HA-nsp12 or Myc-PDCoV N alone served as controls. At 36 h post-transfection, cells were harvested and lysed for Co-IP assays using anti-Myc antibodies, as indicated. (b) HEK293T cells were co-transfected with PDCoV N and nsp12 expression plasmids, and their subcellular localization was analysed by confocal microscopy. (c, d) HEK293T cells were co-transfected with FLAG-pMx1, HA-nsp12 and Myc-PDCoV N expression plasmids. Co-IP assays were performed to evaluate (c) the effect of pMx1 on the interaction between PDCoV N and nsp12, and (d) the influence of nsp12 on the interaction between pMx1 and PDCoV N.

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  Fig. 7.. The predicted interface between pMx1 and PDCoV N is located within the GTPase domain of pMx1. (a) Three-dimensional structural model of the complex formed by the pMx1 monomer and PDCoV N protein, predicted using AlphaFold three and visualized with PyMOL. The pMx1 monomer is shown in dark blue, and PDCoV N is shown in light blue. (b) Enlarged view highlighting the predicted interaction interface between pMx1 and PDCoV N. (c) Structural domain annotations and specific amino acid residues involved in the predicted interaction between the pMx1 monomer and PDCoV N. (d) Schematic representation of the relative positions of the predicted interaction residues within the pMx1 protein. (e–h) WT pMx1 and eight pMx1 point mutants were transfected into IPEC-J2 and IPI-2I cells. At 24 h post-transfection, cells were infected with PDCoV (m.o.i.=0.5). PDCoV N mRNA and protein levels in cell lysates were analysed by RT-qPCR and Western blot. Data are presented as the mean±sd of three independent experiments. Statistical significance was analysed using one-way ANOVA with Dunnett’s post-hoc test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant.