Figure 9

PRRSV nsp4 cleaves GBP1 at E338 and this cleavage impairs the antiviral activity of GBP1. A Sequence logo of the polyprotein junctions cleaved by PRRSV nsp4 from different strains. An amino acid sequence logo of the substrate was generated by using WebLogo. B HEK-239 T cells were co-transfected with Flag-GBP1, -GBP1 mutants and HA-nsp4. At 48 h post-transfection, cell lysates were prepared and analyzed by Western blotting. C Marc-145-GBP1-E338A-flag cells were mock infected or infected with 0.1 MOI of PRRSV. Cells were harvested at 24, 36, 48 hpi and subjected to Western blot analysis. D–F Mutation of nsp4 cleavage site enhanced the antiviral activity of GBP1. Marc-145-Vector, -GBP1-flag and -GBP1-E338A-flag cells were treated with 0.1 MOI of PRRSV. Cells were harvested at 36 h post-infection to determine PRRSV ORF7 mRNA and supernatant viral copies via qPCR, and the expression of N protein was determined by Western blotting. G–I Marc-145-GBP1-flag cells were transfected with 1.0 or 2.0 μg nsp4 expression plasmids for 12 h. Then, cells were inoculated with 0.1 MOI of PRRSV. Cells were harvested, PRRSV ORF7 mRNA and supernatant viral copies were detected by qPCR, N protein was detected by Western blotting. GBP1 guanylate-binding protein 1; nsp4 non-structural protein 4; PRRSV porcine reproductive and respiratory syndrome virus; ORF open reading frame; qPCR quantitative PCR.