Figure 3.

NF-κB activity is affected by ORF6 protein. (a) Interleukin-6 or -IFN-β minimal promoters containing the NF-kB responsive elements only (pIL6-κB and pIFNβ-κB, respectively) were used to drive firefly luciferase expression in HEK-293T cells. Renilla luciferase reporter plasmid was co-transfected as an internal control. Three (n = 3) independent measurements were performed and representative data are presented as mean logarithm values of relative luminescence unit (RLU) ± standard deviations. Significance was evaluated as p < 0.05 (**** p < 0.00001, *** p < 0.0005, ** p < 0.005, * p < 0.05, n.s. = not significant). (b) NF-κB nuclear translocation was evaluated in empty vector (Ctr-) or ORF6 expressing A549 cells. Cellular fractionation was performed as described in the Materials and Methods section and equal amounts of proteins (25 μg) were resolved by polyacrylamide gel. NF-κB movement into the nucleus was evaluated by probing the samples with anti-p65 NF-κB subunit specific antibody. In parallel, ORF6 subcellular localization was assessed in the same samples by anti-HA epitope tag antibody staining. Actin or lamin B1 were stained for cytoplasmic or nuclear loading control, respectively. Original blots are presented in Supplementary Materials. (c) Interleukin-6 promoter (pIL-6) was used to drive firefly luciferase expression in HEK-293T cells in combination with the ectopic expressing of the NF-κB activator IKK-α. Where indicated, empty plasmid (Ctr-) or the viral ORF6 protein expressing plasmid was co-transfected. Renilla luciferase reporter plasmid was used as internal control. Three (n = 3) independent measurements were performed and representative data are presented as mean logarithm values of relative luminescence unit (RLU) ± standard deviations. Significance was evaluated as p < 0.05 (**** p < 0.00001, *** p < 0.0005, ** p < 0.005, * p < 0.05, n.s. = not significant).