PB1-F2 inhibits RIG-I N induced IFN via its C-terminal domain.

<p>(A) IFN secretion was determined via NDV-GFP bioassay. 293T cells were transfected with empty vector (pCAGGS), NS1 or PB1-F2 WT or N66S expressing vectors as well as a RIG-I N expression plasmid. Twenty-four hours post transfection, supernatants were harvested and serial two-fold dilutions of the supernatants were used to overlay Vero cells. Twenty-four hours later, Vero cells were infected with NDV-GFP and images were taken 18 hpi. To quantify the GFP signals, the plate was analyzed by a plate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. (B) 293T cells were transfected as described in (A) with the addition of IFN-β and renilla reporter constructs. Twenty-hour hours post transfection, cells were harvested and luciferase and renilla signals were determined by luminometry. In addition, cells were harvested for Western blot analyses. (C) 293T cells were transfected as described in (A) with the addition of ISRE and renilla reporter constructs. Twenty-hour hours post transfection, cells were treated with 1,000 U/mL of universal type I IFN. Twenty-four hours after IFN treatment, cells were harvested for luminometry and Western blot analyses. (D) 293T cells were transfected and analyzed as described in (B). Besides full-length PB1-F2, following truncation constructs were tested for IFN antagonism activity: the N-terminal domain consisting of amino acids 1–37 (nF2) and the C-terminal domain spanning amino acids 38–87 (cF2). All constructs carried an HA tag and were fused to GFP. All data represent means ± standard deviations of one representative experiment (n = 3). Statistical significance was determined using Student's <i>t</i> test. *, p<0.05; **, p<0.01; ***, p<0.001. All data shown are representatives of at least three independent experiments.