Figure 3

Knockdown of endogenous GBP1 promotes PRRSV replication. A and B Efficiency of endogenous GBP1 knockdown by siRNAs. PAMs transfected with 100 nM siGBP1 targeting different sites of GBP1 or siNC were harvested at 36 h post-transfection. The efficiency of GBP1 knockdown was assessed using A qPCR and B Western blotting. C Viability of PAMs transfected with siGBP1 or siNC was determined using a Cell Counting Kit-8 assay. D and E Knockdown of endogenous GBP1 expression enhances PRRSV replication. PAMs transfected with 100 nM siGBP1 were infected with a 0.1 MOI of PRRSV for 36 h. The expression of PRRSV ORF7 mRNA was assessed via D qPCR, and E N protein was assessed via Western blotting. F Marc-145 cells that were pretreated with or without IFN-γ (10, 50, 100 U/mL) for 12 h were inoculated with PRRSV at a MOI of 0.1. Cells were harvested at 36 h post-infection for the detection of N protein by using Western blotting. G–I GBP1 mediated the anti-PRRSV activity of IFN-γ. Marc-145 cells transfected with 100 nM siGBP1 or siNC for 12 h were treated with or without 50 U/mL IFN-γ for another 12 h, followed by infection with a 0.1 MOI of PRRSV for 24 h. The expression of PRRSV ORF7 mRNA, supernatant viral copies and N protein were analyzed via G and I qPCR and H Western blotting, respectively. J GBP1 was necessary for the regulation of the remodeling of the actin cytoskeleton induced by IFN-γ. Marc-145 cells were transfected with either siNC or siGBP1 as indicated. After 12 h, the cells were either left untreated or treated with IFN-γ (100 U/mL) for 24 h and subsequently stained for actin. GBP1 guanylate-binding protein 1; PRRSV porcine reproductive and respiratory syndrome virus; siRNA small interfering RNA; PAM porcine alveolar macrophage; NC negative control; qPCR quantitative PCR; ORF open reading frame; IFN interferon.