Figure 8

PRRSV nsp4 cleaves GBP1 by its 3C-like serine protease activity. A Porcine alveolar macrophages were infected with 0.1 MOI of PRRSV. Cells were collected at 0, 6, 12, 24, 36, 48 hpi to determine the expression of GBP1 mRNA via quantitative PCR and the expression of GBP1 protein via Western blot (B). C Marc-145-GBP1-flag cells were infected with 0.1 MOI of PRRSV. At 12, 24 and 36 hpi, cells were harvested for Western blot analysis to detect GBP1 and N protein expression. D Marc-145-GBP1-flag cells were infected with PRRSV at a MOI of 0.1, 0.5 and 1.0. At 48 hpi, cells were collected and subjected to Western blotting to detect GBP1 and N protein expression. E The effect of PRRSV proteins on GBP1 expression. HEK293T cells were co-transfected with the plasmids expressing Flag-GBP1 and HA-tagged PRRSV proteins. The cells were harvested at 48 hpt and subjected to Western blotting. F HEK293T cells were co-transfected with 1.0 μg Flag-GBP1 and different concentrations (0, 0.5, 1.0 or 1.5 μg) of HA-nsp4. Cells were collected at 48 hpt and assessed via Western blotting. G) Marc-145 cells mock infected or infected with PRRSV (0.1MOI) were treated with or without MG-132 (5 μM), 3-MA (10 mM) or Z-VAD-FMK (10 μM). At 36 hpi, cells were harvested for Western blot analysis. H The role of cellular protein degradation pathways in the reduction of GBP1 was assessed. HEK293T cells were co-transfected with Flag-GBP1 and HA-Nsp4. At 12 hpt, cells were treated with DMSO control, proteasome inhibitor MG-132 (5 μM), autophagy inhibitor 3-MA (10 mM) or the lysosome inhibitor Z-VAD-FMK (10 μM) for 24 h. The expression of GBP1 and nsp4 were examined by Western blotting. I Effect of the HA-nsp4 and its protease defective mutants on GBP1 expression. HEK293T cells were co-transfected with Flag-GBP1 together with HA-nsp4 or its protease defective mutants expression plasmids for 48 h, followed by Western blot analysis. GBP1 guanylate-binding protein 1; nsp4 non-structural protein 4; PRRSV porcine reproductive and respiratory syndrome virus; hpi hours post-infection; hpt hours post-transfection.