Figure 7

GTPase activity of GBP1 is essential for GBP1-nsp4 interaction. A Schematic representation of the protein domains of GBP1. B Expression of truncated mutants of GBP1. GBP1 truncated mutants Flag-GBP1_1-290 and Flag-GBP1_288-591 or empty vector were transfected into HEK293T cells. Cells were collected and analyzed using rabbit anti-flag pAb. C The N-terminal region of GBP1 interacted with nsp4. HEK293T cells were co-transfected with Flag-GBP1_1-290 and full-length HA-nsp4. Cells were harvested and subjected to co-IP analysis using Protein G Magbeads coated with rabbit anti-flag pAb. D The C-terminal of GBP1 did not interact with PRRSV nsp4. Flag-GBP1_288-591 and HA-nsp4 expression plasmids were co-transfected into HEK293T cells for 48 h. Cells were used for co-IP analysis using beads coated with rabbit anti-flag pAb. E The Marc-145-GBP1_1–290-flag cell line was transfected with plasmid HA-nsp4, and a confocal assay was performed. Scale bar, 10 μm. F Colocalization analysis corresponding to Flag-GBP1_1–290 and HA-nsp4. G The N-terminal domain of nsp4 interacted with GBP1. HEK293T cells were co-transfected with Myc-nsp4_1–69 and Flag-GBP1. Cells were harvested for co-IP analysis using beads coated with mouse anti-Myc mAb. H and I HEK293T cells were co-transfected with Flag-GBP1-K51A or -R48A together with HA-nsp4, respectively. At 24 h post-transfection, cell lysates were collected for co-IP detection with beads conjugated with rabbit anti-flag pAb. GBP1, guanylate-binding protein 1; nsp4 non-structural protein 4; co-IP co-immunoprecipitation; PRRSV porcine reproductive and respiratory syndrome virus.