Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
Fig 4
Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production.
(A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A. (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; ****P < 0.0001, **P < 0.01 compared with Mock (B), *P < 0.05 compared with shCtrl (C). (D) Two clones of the FEN1+/− Hep38.7-Tet cells (#1 and #2 in Fig 2) were transduced with pResQ lentiviral vectors carrying FEN1 shRNA. wt, shCtrl. Hep38.7-Tet cells were transduced with the pResQ mock vector. After puromycin selection, FEN1 protein, cytoplasmic NC-DNA, and Hirt-extracted HBV DNA were analyzed by Southern blotting. 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 the FEN1 wt cell line with control shRNA is considered 100%.