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. 2022 Aug 17;14(6):1177–1198. doi: 10.1016/j.jcmgh.2022.08.002

Figure 1.

Figure 1

Establishment of recombinant cccDNA mimicking the cccDNA minichromosome. (A) Schematic diagram of MC-HBV recombination in E coli and MC-HBV drove the entire cycle of pgRNA-rcDNA-cccDNA in hepatocytes. The 5'/3'-intron is represented as a red arrow, the HBV genome is represented as a blue box. (B) EcoRI digestion and gel electrophoresis analysis were performed to confirm the recombination efficiency of MC-HBV induced by arabinose in E coli cells (pmini-HBV PP ≈ 7.3 kb, MC-HBV ≈ 3.3 kb). (C) MC-HBV was transfected into Huh7 cells for 72 hours, PP and pCMV-Cre/parent rcccDNA (prcccDNA) as control, pgRNA/S messenger RNA (mRNA) and cccDNA were detected by RT-PCR and PCR, respectively, using primers P1/P2 as indicated with arrows. MC-HBV/unspliced viral transcripts are 431 nt, wild cccDNA/spliced viral transcripts with a chimeric intron are 303 nucleotides (nt). (D) PP and MC-HBV were transfected into Huh7 cells for 72 hours. HBsAg, HBeAg, and HBc expression were measured by enzyme-linked immunosorbent assay (ELISA), Western blot, and indirect immunofluorescence assay (IFA), respectively (n = 2). (E) MC-HBV was transfected into Huh7 cells, and persistent HBV replication was dynamically monitored by detecting HBsAg/HBeAg expression (ELISA) and nascent cccDNA formation (PCR). The ZHX2 gene of undigested whole-cell genomic DNA was amplified as the DNA loading control. (F) MC-HBV was transfected into Huh7 cells for 5 days, then virion particles in the supernatant were concentrated to infect Huh7NTCP cells for 5 days; HBsAg and HBeAg were detected by ELISA and IFA assay; and HBV-DNA, viral pgRNA, and cccDNA were analyzed by PCR (n = 3). (G) HepG2 cells were transfected with MC-HBV, and HepG2NTCP cells were infected with HBV at 200 genome equivalents (Geq) for 4 days, and the enrichment of active makers CBP, p300, H3K4me3, H3K27ac, and H3K36me3, and repressive makers H3K9me3 and H3K27me3, on cccDNA was measured by ChIP assay (n = 3). (H) HA-HBx and MC-HBV were cotransfected into Huh7 cells for 72 hours, or (I) MC-HBV was transfected Huh7 cells for 24 hours and then treated with 2000 U/mL IFN-α for another 48 hours. ChIP assay was performed to evaluate the occupancy of acetylation of histone H4 (AcH4) on cccDNA, and HBsAg/HBeAg expression and pgRNA and interferon stimulated exonuclease gene 20 (ISG20) mRNA levels were detected (n = 3). Data are presented as means ± SD. ∗P < .05, ∗∗P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.