FIG 1.

FoxO4 overexpression inhibits cccDNA-mediated transcription and HBV replication. (A to G) PrcccDNA/Cre was transfected into Huh7 cells together with control empty vector or increasing amount of FoxO4-Flag for 48 or 72 h. (A) The protein level of FoxO4-Flag in prcccDNA/Cre-transfected Huh7 cells was determined by Western blotting. The levels of HBsAg (B), HBeAg (C), HBV RNAs (D), and preC-pgRNA (E) were determined by ELISA and qRT-PCR, respectively. The levels of HBV DNA were determined by qPCR (F) and Southern blotting (G). (H to K) Linear HBV monomers were transfected into Huh7 cells together with control empty vector or increasing amounts of FoxO4-Flag for 48 h, and the levels of FoxO4-Flag, HBsAg, HBeAg, preC-pgRNA, and HBV DNA were determined as in panels A to G. cccDNA was extracted by Hirt’s method as described in Materials and Methods and then subjected to quantification by qPCR (K). (L to N) HepG2-NTCP cells were electrotransfected with FoxO4-Flag for 48 h and then infected with HBV at 10³ vge/cell as described in Materials and Methods. The levels of FoxO4-Flag, cccDNA (at day 3 postinfection) and the levels of HBsAg, HBeAg, preC-pgRNA, and HBV DNA (at day 9 postinfection) were determined as described above. The data show means ± the SD of triplicates and are representative of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significance; RI, replicative intermediates).