IFI16 represses viral gene transcription from a recombinant (r)cccDNA template of HBV. (A) Diagram, modified from Qi et al. [22], illustrates the generation of rcccDNA via Cre/loxP recombination. (B) PCR quantification of rcccDNA with P1/P2 primers using Hirts extracts of Huh7 cells transfected with indicated plasmids for 72 h. Data represent the average of two independent experiments. (C) Immunofluorescence microscopy of Huh7 cells co‐transfected for 72 h with prcccDNA and pCMVGFP‐Cre, with (+) or without (−) pCMV‐FLAG‐IFI16. Arrows indicate cells expressing IFI16 (blue), co‐staining with GFP‐Cre expressing cells, and HBc (red) expressing cells. (D–F) Immunofluorescence microscopy of Huh7 cells co‐transfected for 72 h with prcccDNA and pCMVGFP‐Cre, with (+) or without (−) pCMV‐FLAG‐IFI16. (E) Quantification of HBc/Cre and (F) GFP‐Cre‐positive cells, with (+) or without (−) IFI16 expression. Data are expressed as mean ± standard error of the mean (SEM) from n = 3. **p < 0.01 and ns = not significant. (G) qRT‐PCR quantification of viral RNAs (preC/pgRNA and total HBV RNAs) and IFI16 mRNA using RNA isolated from Huh7 cells, transfected with indicated plasmids for 72 h. Data are expressed as mean ± SEM from three independent experiments. **p < 0.01.