Fig 1.
Schematic illustrations of HBV rcDNA, terminal redundancy (TR or r), and cccDNA.
(A) The positive (+) strand and negative (-) strand of rcDNA are labeled. The broken line indicates varying lengths of (+) strand DNA. The RNA primer at the 5’ end of (+) strand is depicted with a curved line. Viral polymerase (Pol) covalently attached to the 5’ end of (-) strand is indicated by a solid dot. DR1 and DR2 motifs are shown as gray rectangles. The mandatory molecular reactions required for converting rcDNA into cccDNA are listed. (B) A sequence fragment (nt 1810–1840) flanking the TR region (nt 1821–1828, shown in blue letters) of the genotype D HBV genome (GenBank Accession No.: U95551.1) is displayed. Depending on the base pairing between (+) strand DNA and 3’ or 5’ TR, a DNA flap stucture forms on the 5’ or 3’ end of (-) strand DNA of rcDNA, respectively.
Fig 2.
Establishment of HepDES-C1822G cell line.
(A) Mutagenesis strategy for introducing a point mutation G1822C into the 3’ TR of rcDNA. HBV pgRNA is schematically illustrated, with the DR1/DR2 motifs and epsilon (ε) stem-loop structure being indicated. The C1822G mutation (in red) is introduced into the 5’ end of pgRNA coding sequence, which will be reverse transcribed into 1822C (in red) in the 3’ TR of (-) strand DNA, while the 5’ TR maintains a wild type 1822G. (B-C) HepDES19 cell line and the established HepDES-C1822G cell line were uninduced (+ tet) or induced (- tet) for 14 days. The tet-inducible (tet-off) HBV core DNA replication (B) and cccDNA formation (C) were analyzed by Southern blot. (D) HBV core DNA from the induced HepDES19 and HepDES-C1822G cells were subjected to (-) strand DNA 3’ RACE and Sanger sequencing. The RACE anchor-HBV 3’ TR junction sequences are labeled on the sequence chromatograms. Arrows indicate the wt 1822G and mutant 1822C in the 3’ TR of HBV (-) strand DNA from induced HepDES19 and HepDES-C1822G cells, respectively.
Fig 3.
NGS analyses of HBV TR sequences in HepDES19 and HepDES-C1822G cells.
(A) Selection of TR template for (+) strand DNA synthesis. The cytoplasmic HBV core DNA was extracted from the induced HepDES19 and HepDES-C1822G cells and subjected to (+) strand-specific PCR amplification of a 458-bp fragment containing the complementary sequence of TR region (S1A Fig). The PCR amplicon was sequenced by NGS and the nucleotide constitution at each position within nt 1812–1825 was plotted. The column of nt 1822 nucleotide constitution is marked with an asterisk. (B) Selection of TR for retention on cccDNA. Total Hirt DNA extracted from the induced HepDES19 and HepDES-C1822G cells were subjected to heat denaturation and PSAD treatment to remove DP-rcDNA, followed by PCR amplification of a 211-bp cccDNA fragment flanking the TR region (S1B Fig). NGS of the PCR amplicon was performed, and the nucleotide constitution at each position within nt 1812–1825 of both strands was plotted. Asterisks mark the columns representing nucleotide constitution at nt 1822 on both (+) and (-) strands of cccDNA.
Fig 4.
XPF knockout has no influence on HBV cccDNA production.
(A) Verification of XPF knockout. The XPF gene editing and protein expression in HepAD38 control K.O. cells and XPF K.O. cell clone 5 (c5) and 13 (c13) were analyzed by T7E1 assay and Western blot, respectively. β-actin served as Western blot loading control. (B) HepAD38 control and XPF K.O. cells were cultured in tet-free medium for 14 days. HBV cytoplasmic core DNA and total Hirt DNA were extracted and detected by Southern blot. A 3.2 kb HBV DNA served as size marker. Prior to gel loading, the Hirt DNA samples were heat denatured to convert DP-rcDNA into ssDNA, followed by EcoRI digestion to linearize cccDNA into the unit-length dslDNA. The band intensities of cytoplasmic rcDNA and nuclear cccDNA were quantified and their relative levels were expressed as percentage (%) of control samples. (C) A portion of the Hirt DNA samples were heat denatured and digested by PSAD to remove DP-rcDNA. The remaining cccDNA was quantified by qPCR and normalized by total Hirt DNA qPCR. The relative cccDNA levels were plotted (control was set to 1) (mean ± SD, n = 3; ns: not significant).
Fig 5.
Mus81 knockout reduces HBV cccDNA production.
(A) Verification of Mus81 knockout. Mus81 gene editing and protein expression in HepAD38 control K.O. cells and Mus81 K.O. clone 1 (c1), clone 7 (c7), and clone 8 (c8) were verified by T7E1 assay and Western blot, respectively. (B-C) HepAD38 control and Mus81 K.O. cells were induced in tet-free medium for 14 days, followed by (B) HBV core DNA and cccDNA Southern blot analyses and (C) cccDNA qPCR assay (mean ± SD, n = 3; ***p<0.001).
Fig 6.
Mus81 knockout inhibits de novo cccDNA formation in HBV infection system.
HepG2-NTCP control and Mus81 K.O. cells were transfected with control vector or plasmid expressing Flag-tagged Mus81. Two days post-transfection, the cells were infected with HBV at MOI of 200 for 5 days and subjected to the following analyses. (A) Protein expression of Mus81 was analyzed by Western blot with β-actin serving as loading control, and cccDNA was detected by Southern blot with mitochondrial DNA (mtDNA) serving as loading control. (B) cccDNA qPCR (mean ± SD, n = 3; ***p<0.001). (C) HBc immunofluorescence assay. Cell nuclei were counterstained with DAPI. The percentage of HBc-positive cells is indicated. Scale bar: 100 μm.
Fig 7.
Mus81 specifically cleaves TR in a 3’ flap configuration in vitro.
(A) Schematic illustration of the structural and catalytic domains of human Mus81 and Eme1. Two Mus81 enzymatically inactive mutations (T383R and A387R) are indicated. HhH: helix-hairpin-helix domain. (B) The Mus81 (aa 246–551)/Eme1 (aa 178–570) enzymatic domain complex was expressed in E. coli., purified, and verified by SDS-PAGE. (C) Schematic presentation of the fluorescence-labeled synthetic HBV 3’ TR-flap DNA substrate (referred as substrate 1). The green star indicates the fluorescent label. (D) DNA substrate 1 was left untreated or treated with varying concentrations of Mus81-Eme1, followed by native acrylamide gel separation and fluorescence image scanning. (E) DNA substrate 1 was left untreated or treated with wt or mutant Mus81 (T383R+A387R)-Eme1 and subjected to in-gel fluorescence analysis. (F) Schematic presentation of the fluorescence-labeled synthetic HBV 5’ TR-flap DNA substrate (referred as substrate 2). (G) DNA substrate 2 was left untreated or treated with wt Mus81-Eme1, followed by in-gel fluorescence analysis.
Fig 8.
Knockout of FEN1 reduces HBV cccDNA production.
(A) Verification of FEN1 knockout. FEN1 gene editing and protein expression in HepAD38 control and FEN1 K.O. cells were analyzed by T7E1 assay and Western blot, respectively. (B-C) HepAD38 control and FEN1 K.O. cells were induced in tet-free medium for 14 days, followed by (B) HBV core DNA and cccDNA Southern blot analyses and (C) cccDNA qPCR assay (mean ± SD, n = 3; **p<0.01).
Fig 9.
Knockout of XPG has no effect on HBV cccDNA formation.
(A) Verification of XPG knockout. XPG gene editing and protein expression in HepAD38 control and XPG K.O. cells were analyzed by T7E1 assay and Western blot, respectively. (B-C) HepAD38 control and XPG K.O. cells were induced in tet-free medium for 14 days, followed by (B) HBV core DNA and cccDNA Southern blot analyses and (C) cccDNA qPCR assay (mean ± SD, n = 3; ns: not significant).
Fig 10.
Knockout of Mus81 or FEN1 reduced CM-rcDNA production.
(A) Diagram of HBV DNA species and host mitochondrial DNA (mtDNA) in Hirt DNA samples and their remaining products after ExoI/III digestion. DP-rcDNA can be completely removed by ExoI/IIII. CM-rcDNA will become a single-stranded circle (-) strand DNA after ExoI/III treatment. HBV cccDNA and host mtDNA will survive ExoI/III digestion. (B) HepAD38 control, Mus81, and FEN1 K.O. cells were induced in tet-free medium for 10 days. The harvested cells were subjected to HBV cytoplasmic core DNA and total cellular Hirt DNA extraction, and Hirt DNA samples were further treated with ExoI/III, followed by Southern blot analyses.
Fig 11.
Mus81 and FEN1 are involved in HBV infection in PHH cells.
PHH cells seeded in 6-well plate were transfected with 100 pmol of si-control, si-Mus81, si-FEN1, or si-Mus81&si-FEN1 (100 pmol each) for 2 days, followed by HBV infection (500 MOI) for additional 3 days. The harvested PHH cells were subjected to analyses as follows. (A) Western blot of Mus81, FEN1 and loading control β-actin. HepAD38 control, Mus81, and FEN1 KO cell samples served as controls. (B) RT-PCR detection of Mus81, FEN1, and GAPDH mRNA. PCR without the prior RT step served as control to rule out a possibility of genomic DNA contamination. (C) Immunofluorescence of intracellular HBcAg. Cell nuclei were counter staining by DAPI. The percentage of HBcAg-positive cells is indicated. (D) qPCR of intracellular cccDNA. The relative levels of cccDNA were plotted (mean±SD, n = 3; ***p<0.001, ****p<0.0001). (E) Supernatant HBeAg CLIA (mean±SD, n = 2; *p<0.05, **p<0.01).
Fig 12.
Identification of potential FEN1 and Mus81 cleavage sites in TR by RACE-NGS.
(A) Schematic illustration of the cohesive region of HBV rcDNA. The (+) and (-) strands of rcDNA and their 5’ and 3’ ends are denoted. The TR (r) nucleotide sequences of (-) strand DNA are shown in blue. For convenience, the 3’ TR is shown as complementary with (+) strand DNA. The bracketed small T indicates the noncanonical first primed nucleotide 1829T at the 5’ end of (-) strand DNA. The nucleotide (nt) positions of genotype D HBV DNA are indicated according to the Galibert nomenclature [78]. DR1 and DR2 regions are boxed. The parallel dotted lines represent the omitted internal DNA sequences between TR and DR2. The 5’ RNA primer sequences are shown in red lowercase letters, the tilted sequences indicate the 5’ uncomplementary portion. (B-C) The nuclear DP-rcDNA was extracted from induced HepAD38 control, FEN1, and Mus81 K.O. cells and subjected to (-) strand DNA 5’ and 3’ RACE-NGS assay, followed by bioinformatic analyses to identify the terminal nucleotide within the TR sequence. The percentage of the number of each full-length and truncated 5’ and 3’ end, relative to the total 5’ and 3’ TR RACE-NGS counts for each sample, was plotted, respectively. The Y-axis represents the terminal nucleotide of identified TR with varying lengths. The terminal nucleotide of full-length 5’ and 3’ TR is marked with an asterisk. Arrows indicate the cleavage sites for FEN1 (B) and Mus81 (C).
Fig 13.
Model of HBV rcDNA TR processing during cccDNA formation.
(A) Diagram of a proposed molecular pathway of cccDNA formation with DP-rcDNA as an intermediate. For convenience, the 5’ TR (r) on the (-) strand of rcDNA/DP-rcDNA is depicted as a flap structure. In the cytoplasm, the gap filling of (+) strand DNA of rcDNA triggers the removal of viral RNA primer from 5’ end of (+) strand and Pol from 5’ end of (-) strand DNA, giving rise to the cytoplasmic DP-rcDNA, followed by import of DP-rcDNA into nucleus, where the cellular DNA repair machinery removes one copy of the TR on (-) strand DNA of DP-rcDNA and prepares the termini of both DNA strands for ligation into cccDNA [19,25–27,31]. (B) The proposed model of TR processing on the (-) strand DNA of nuclear DP-rcDNA presents four major possible scenarios, depending on the different polarities of the flap structure formed by TR and the timing of FEN1 and Mus81 involvement. Refer to the text for a detailed description.