FIG. 4.

NS5A expression is associated with increased IL-8 protein production and inhibition of the antiviral effects of IFN. (A) Correlation among levels of IL-8 in culture supernatants, amounts of NS5A protein expression, and levels of EMCV rescue in the trans rescue assay. HeLa cells expressing FL-NS5A-NR were grown for 48 h in medium containing 0, 0.001, 0.01, and 1.0 μg of tetracycline per ml for 48 h, treated with 20 U of IFN per ml for 24 h, and infected with EMCV at an MOI of 0.01 for 24 h. The amount of IL-8 protein in culture supernatants, determined by ELISA, is indicated in the bar graph. Changes in EMCV titers were determined 24 h postinfection by viral plaque assay. The levels of NS5A protein expression were determined by Western blot analysis of equal amounts of total cellular protein extracts and are presented as +++, ++, +, and −, which correspond to high, intermediate, low, and undetectable levels of NS5A protein, respectively, as determined by computerized scanning of the chemiluminescent signal. Fold EMCV rescue represents the difference in EMCV titers in the presence of IFN among cells treated with 0, 0.001, and 0.01 μg of tetracycline per ml versus the 1.0-μg/ml concentration. (B) IL-8 inhibits the antiviral actions of IFN in vitro. HeLa Tet-Off cells were pretreated with or without 33 ng of recombinant human IL-8 per ml for 6 h and then with 20 U of IFN per ml for 18 h. Cells were then infected with EMCV at an MOI of 0.1 for 24 h. Supernatants were harvested, and the amount of EMCV was determined by titration on L929 cells. Error bars represent standard deviations, and P values derived from Student's t tests are indicated.