Intrinsic and innate immune properties of PMLIV.

<p>(A) U373MG cells were left untreated or treated for 24 h with 100 units/ml of IFN-β in the absence or the presence of 20 µg/ml of purified anti-IFNAR1 mAb before infection with VSV at an MOI of 1 for 8 h (left panel). U373MG and U373MG-PMLIV cells were infected with VSV at an MOI of 0.2 for 8 h (VSV¹) or 12 h (VSV²) in the absence or the presence of 20 µg/ml of anti-IFNAR1 mAb (right panel). Cell extracts were analyzed by Western blotting and revealed by antibodies directed against VSV, STAT1 or Actin. (B) Activity of secreted IFN in infected U373MG-PMLIV cells. Culture supernatants (from U373MG-EV cells [S_EV] and U373MG-PMLIV cells [S_PMLIV]) were treated with acid buffer to inactivate virus, and the pH was then neutralized. One series of HeLa cells were left untreated as a control (Ctrl) or treated for 24 h with 100 units/ml of IFN-β, S_EV or S_PMLIV, then their extracts were analyzed by Western blot for PKR and STAT1 (left panel). The second series of HeLa cells were infected with VSV at an MOI of 0.2 for 8 h and the cell extracts were analyzed by Western blotting using anti-VSV and anti-Actin antibodies (right panel). (C) Secreted IFN activates STAT1 phosphorylation (left panel). HeLa cells were left untreated (Ctrl) or treated for 30 min with 1000 units/ml of IFN-β, S_EV or S_PMLIV, then their extracts were analyzed by Western blot for P-STAT1 and STAT1. Quantification of produced type I IFN upon VSV infection (right panel). IFN was quantified using the reporter cell line HL116 that carries the luciferase gene under the control of the IFN-inducible 6–16 promoter <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003975#ppat.1003975-Uze1" target="_blank">[55]</a>. HL116 cells were incubated for 8 h with a standard containing titrated human IFN-β, the supernatants from U373MG-EV [S_EV] and U373MG-PMLIV [S_PMLIV] cells infected with VSV at an MOI of 1 for 20 h or the supernatants from uninfected U373MG-EV and U373MG-PMLIV. Cells were then lysed and luciferase activity measured. IFN levels were expressed as equivalent of IFN-β concentration, in IU/ml. Means and standard deviations of two independent experiments are shown.