Figure 6

PRRSV nsp4 interacts with GBP1. A and B Exogenous co-IP assay in HEK293T cells. HEK293T cells were co-transfected with Flag-GBP1 (1 μg) with or without HA-nsp4 (1 μg). At 48 h post-transfection, cells were harvested for co-IP analysis using Protein G Magbeads coated with A rabbit anti-flag pAb (green) or B mouse anti-HA mAb (red). C Expression plasmids Flag-GBP1 and HA-nsp4 were co-transfected into HEK293T cells and then subjected to a confocal assay. D and E Marc-145-Vector or Marc-145-GBP1-flag cells were infected with PRRSV at a MOI of 1.0 for 48 h. Cells were harvested and subjected to co-IP assay using beads coated with D rabbit anti-flag pAb or E mouse anti-nsp4 pAb. F Marc-145-Vector or Marc-145-GBP1-flag cells infected with PRRSV (MOI = 0.1) for 36 h were fixed and stained with rabbit anti-flag pAb (green) and mouse anti-nsp4 pAb (red), and the nuclei were stained with DAPI. Fluorescent images were acquired with a confocal laser scanning microscope. Scale bar, 10 μm. GBP1 guanylate-binding protein 1; PRRSV porcine reproductive and respiratory syndrome virus; nsp4 non-structural protein 4; co-IP co-immunoprecipitation.