Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
Fig 2
FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production.
(A–D) Hep38.7-Tet cells were transfected with FEN1-specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *P < 0.05, **P < 0.01, ***P < 0.001 compared with siCtrl. (E–G) The pX330-FEN1 plasmid, which expresses Cas9 mRNA and sgRNA for exon 2 of the FEN1 gene, was transfected with a blasticidin-resistant gene expression vector into Hep38.7-Tet cells. After drug selection, two resistant clones (#1 and #2) were analyzed. (E) RT-qPCR and Western blotting analyses of FEN1 expression levels. (F) Efficiency of cccDNA formation. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *P < 0.05, **P < 0.01 compared with parental groups. (G) Southern blot analysis of cytoplasmic NC-DNA and Hirt-extracted HBV DNA. Hirt-extracted HBV DNA was analyzed with or without heat treatment and subsequent EcoRI digestion. Densitometric analysis of the EcoRI-digested cccDNA signal is shown below the Southern blot images. The signal for parental cell line is taken as 100%.