HBV Pol suppresses NF-κB activity in HEK 293 T, HepG2, and Huh7 cells.

<p><i>A</i>. Western blot analysis revealed the expression of the HBV Pol in HEK 293 T (lanes 1 and 2), HepG2 (lanes 3 and 4), and Huh7 (lanes 5 and 6) cells. Cells were transfected with 0.8 µg of pAHC-Pol expressing the HBV Pol (lanes 1, 3, and 5) or 0.8 µg of empty vector (pAHC) as a negative control (lanes 2, 4, and 6). At 32 h posttransfection, expression of the plasmids was determined by western blot using a mouse anti-HA antibody. <i>B</i>. HepG2 and Huh7 cells were cotransfected with 0.1 µg of pNF-κB-Luc (5×NF-κB binding site promoter-driven luciferase reporter plasmid) and 10 ng of pTK-Renilla-Luc along with 0.6 µg of pAHC-Pol or 0.6 µg of pAHC-X as a positive control and normalized to the pAHC empty vector. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. <i>C</i>. 293 T cells were cotransfected with 0.1 µg of 5×pNF-κB-Luc and 25 µg of pTK-Renilla-Luc along with different amounts of pAHC-Pol (lane 3, 0 ng; lane 4, 200 ng; lane 5, 400 ng; lane 6, 600 ng). The total amount of plasmid was adjusted with the empty vector (pAHC) to 600 ng. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. <i>D</i>. HepG2 cells were cotransfected with 100 ng of 5×pNF-κB-Luc and 25 ng of pTK-Renilla-Luc together with 100 ng of the activating plasmid, pRK-X (where X is TRAF2, TRAF6, IKKα, or IKKβ), or the corresponding empty vector, pRK, along with 600 ng of pAHC-Pol or 600 ng of the pAHC empty vector as indicated below the graph. After 24 h of transfection, relative luciferase activities were determined. <i>E</i>. HepG2 cells were transfected with empty vector, WT, X-null, E-null, E/X-null and P-null HBV replicon together with the 5×pNF-κB-Luc reporter construct and pTK-Renilla-Luc, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. <i>F</i>. HepG2.2.15 and HepG2 cells were transfected with 100 ng of 5×pNF-κB-Luc and 25 ng of pTK-Renilla-Luc. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. <i>G</i>. HepG2 cells were transfected with increasing doses of the HBV Pol expression construct, along with the IFNβ luciferase reporter construct and TLR3 expression construct. Cells were stimulated with 25 mg/mL poly I:C directly added to the medium for 8 h. Luciferase activities correspond to the average of results from at least three independent experiments, and data are shown as means ± SE (**p<0.01, *p<0.05).