FIG. 5.

The TTV ORF2 protein suppresses NF-κB activity in the noncanonical NF-κB pathway. (A) HeLa, HepG2, and RAW264.7 cells were cotransfected with 0.1 μg of pcDNA3.1(+)-NIK or pcDNA3.1(+)-dnNIK, 0.1 μg of pNF-κB-Luc, 10 ng of tk-Renilla-Luc along with 0.6 μg of pcDNA3.1(−)-TTV2, or 0.6 μg of pcDNA3.1(−) as indicated below the graph. At 48 h posttransfection, cells were harvested for luciferase assay. Luciferase activities correspond to averages of results from at least three independent experiments, and data are means ± SE (*, P < 0.05). (B) HeLa cells were transfected with 0.1 μg of pcDNA3.1(+)-NIK and 0.6 μg of either the empty vector pcDNA3.1(−) or pcDNA3.1(−)-TTV2 as indicated below the graph. At 48 h posttransfection, cytoplasmic and nuclear fractions were prepared as described in Materials and Methods. Both cytoplasmic proteins and nuclear proteins were analyzed by Western blotting with anti-p100/p52 and anti-Rel-B antibodies to reveal the localization of NF-κB subunits. Nucleus-specific anti-YY1 antibody was used as a control. Results are representative of three different experiments.