FIG. 2.

NS1 of A/Tx/36/91 mediates a general inhibition of gene expression. (A) Schematic representation of functional domains in the NS1 protein, showing the RNA-binding domain (positions 1 to 73) (40), the eIF4G-binding domain (positions 81 to 113) (1), the CPSF interaction domain (around position 184) (51), and the PABII-binding domain (positions 223 to 230) (12). Numbers indicate amino acid positions. 293T (B, E, F, and I) and Vero (C, D, G, and H) cells were cotransfected with reporter plasmids carrying the FF-Luc gene under the control of the IFN-β promoter (B and F) or the Mx1 promoter (C and G) and REN-Luc under the control of the constitutive SV40 promoter (D and H), together with expression plasmids encoding HA-tagged versions of NS1, an NS1 chimera of A/PR/8/34 and A/Tx/36/91, or C-terminally truncated Tx NS1 constructs or with an empty plasmid (ctrl). At 16 h posttransfection, the cells were infected with SeV (B and F) or treated with 200 U/ml of IFN-α2a (C and G) for 16 h and then analyzed for reporter gene expression. (E and I) Lysates of 293T cells were analyzed for NS1 protein expression by Western blotting using an HA-specific antiserum. The numbers indicate the relative intensities of the NS1 protein bands, with PR NS1 activity defined as 100. The data from one representative experiment are shown.