FIG 4.

Mutations in the GKYLQRRLQ motif attenuate the ability of PRRSV to suppress the expression of IFN-α and ISGs. Porcine alveolar macrophages seeded into a 24-well plate were infected with the WT virus or nsp1β mutants at an MOI of 1.0, and cell culture supernatants were harvested at 12 h postinfection. (A) IFN-α production was quantified by using a ProcartaPlex Porcine IFN alpha Simplex kit (eBioscience, San Diego, CA). Each data point shown represents the mean value from three independent experiments with duplicates, and error bars show SEM. (B) The mRNA expression level of ISG15 was evaluated by quantitative real-time RT-PCR and normalized to the level of endogenous β-tubulin mRNA. (C) The mRNA expression level of IFIT1 was evaluated by quantitative real-time RT-PCR and normalized to the level of endogenous β-tubulin mRNA. (D) The mRNA expression level of IFITM1 was evaluated by quantitative real-time RT-PCR and normalized to the level of endogenous β-tubulin mRNA. Values in panels B to D are expressed as means ± SEM from three independent experiments. (E) The expression level of nsp1β at 12 h postinfection was determined by Western blot analysis with nsp1β-specific MAb 123-128. β-Tubulin was detected as a loading control. The membrane was incubated with a mixture of primary MAb 123-128 and MAb against β-tubulin. The secondary antibody IRDye 800CW goat anti-mouse IgG(H+L) (Li-Cor Biosciences, Lincoln, NE) was used to visualize the target proteins with a digital image system (Odyssey infrared imaging system; Li-Cor Biosciences, Lincoln, NE). Statistical significance between the wild-type virus-infected group and mutant virus-infected groups was determined by one-way ANOVA and Tukey's test and is indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001).