Figure 1.

Ability of master donor virus (MDV) A/AA/6/60 live attenuated influenza vaccine (LAIV) PA and NS1 proteins to block host gene expression: Human 293T cells (5 × 10⁵, 12-well plates, triplicates) were transiently co-transfected, using LPF2000, with expression plasmids encoding GFP and Gluc under the control of a chicken beta actin promoter (pCAGGS GFP and pCAGGS Gluc, respectively) together with pDZ plasmids encoding wild-type (WT⁺) or mutant (MT⁻) PA or NS1 proteins; or empty (E) plasmid as control. At 24 h post-transfection (p.t.), cells were analyzed by GFP expression (A,D) under a fluorescent microscope and by Gluc activity (B,E) from tissue culture supernatants (TCS) using a luminometer. Representative images are shown. Scale bar = 100 μm. Results represent the means and SDs of triplicate values. Protein expression from cell lysates was evaluated by Western blot (C,F) using specific antibodies for PA (C) or NS1 (F), or actin as the loading control. Molecular markers are indicated on the left. Western blots were quantified by densitometry using the software ImageJ. Relative band intensities (as described in Materials and Methods) are indicated. ns, not statistical.