FIG. 3.

Suppression of IFN-dependent activation and nuclear translocation of STAT-1. (A) Vero cells stained for pSTAT-1. Cells treated with 2,000 U/ml of IFN-β demonstrate the nuclear location of activated STAT-1, pSTAT-1 (+IFN-β). ZEBOV VP24, used as a positive control, inhibits nuclear translocation of pSTAT-1 in IFN-treated Vero cells at 1 day posttransfection but is unable to inhibit activation of STAT-1, as evidenced by cytoplasmic detection of STAT-1 in cells expressing viral protein (merge). α, anti. (B) Vero cells were transfected with plasmids expressing ANDV NP or GPC. At 1day posttransfection, cells were treated with 2,000 U/ml of IFN-β for 15 min, fixed, and stained using antibodies detecting ANDV nucleocapsid protein or Gn and Gc proteins (top left panels) and pSTAT-1 (top right panels). The corresponding panels below show the merged images. (C) Vero cells were transfected with plasmids expressing ANDV Gn or Gc. At 1 day posttransfection, cells were treated with 2,000 U/ml of IFN-β for 15 min, fixed, and stained using antibodies detecting ANDV Gn or Gc protein (left) and pSTAT-1 (middle). The merged images are shown at the right. Arrows indicate inhibition of STAT-1 activation and subsequent nuclear translocation; arrowheads indicate cells in which STAT-1 activation and nuclear translocation were not inhibited. (D) Flow cytometry investigating the effect of V5-tagged viral protein on STAT-1 activation using LGTV NS5 as a positive control confirms inhibition of STAT-1 activation by ANDV NP.