FIG. 6.

TTV ORF2 protein inhibits the expression of NF-κB-mediated downstream genes through its effect on NF-κB. (A) HeLa and HEK293 cells were cotransfected with 0.1 μg of pGL3 X-p-Luc or pGL3 X-p-Luc (ΔNF-κB) (where X is COX-2, IL-6, or IL-8) and 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 treated with LPS (1 μg/ml) for 6 h and then harvested for luciferase activity. Luciferase activities correspond to averages from at least three independent experiments, and data shown are means ± SE (*, P < 0.05). (B) RAW264.7 cells were transfected with 0.6 μg of pcDNA3.1(−)-TTV2 or 0.6 μg of pcDNA3.1(−). At 48 h posttransfection, cells were stimulated with LPS (1 μg/ml) or left unstimulated for 6 h as indicated. The RT-PCR products of COX-2, IL-6, or IL-8 mRNA were detected by agarose gel electrophoresis. GAPDH was used as a control. IL-6, IL-8, and COX-2 mRNAs were measured by gel analysis software and normalized by calculating the ratio of mRNA to GAPDH. (C) RAW264.7 cells were transfected with 0.6 μg of the empty vector pcDNA3.1(−) without stimulation as a control or with different amounts of pcDNA3.1(−)-TTV2 (lane 2, 0 ng; lane 3, 200 ng; lane 4, 400 ng; lane 5, 600 ng) and treated with LPS (1 μg/ml) for 6 h. Cell extracts were prepared, and the protein amounts were determined using anti-COX-2 antibody. (D) RAW264.7 cells were transfected with 0.6 μg of pcDNA3.1(−)-TTV2 or 0.6 μg of pcDNA3.1(−). At 48 h posttransfection, cells were stimulated with LPS (1 μg/ml) or left unstimulated for 6 h as indicated below the graph. IL-6 and IL-8 in the culture supernatant of RAW264.7 cells were measured using the ELISA method. Results are representative of three different experiments.