Fig 9. Protein C6 inhibits ISRE-dependent gene expression during VACV infection.

(A) Multiple wells (n = 6 per condition) of a 96-well plate containing HEK293T cells were transfected with plasmids expressing firefly luciferase under the control of an ISRE promoter and a plasmid constitutively expressing renilla luciferase. Sixteen h post transfection, cells were either mock-infected or infected with vΔC6 or wt VACV at 5 PFU/cell and 5 h later the cells were harvested and the expression of firefly luciferase was measured and normalised to renilla luciferase expression. ****P<0.0001. (B) Lysates from cells treated as in (A) were subjected to SDS-PAGE and immunoblotting for VACV proteins D8 and C6, and for cellular GAPDH. (C) HEK293T cells were transfected by the same plasmids as described in (A) and 16 h later the cells were transfected with 100 ng/well (96-well plate) of a plasmid expressing IRF9-S2C for 4 h. The cells were then infected with vΔC6 or wt VACV at 5 PFU/cell for 5 h. The expression of firefly luciferase was measured and normalised to renilla luciferase expression. **P<0.01. (D) Lysates from cells treated as in (C) were analysed by SDS-PAGE and immunoblotting as in (B). In (B) and (D) the positions of molecular mass markers are shown in kDa on the left.