Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

<div><p>Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An <i>in vitro</i> FEN activity assay demonstrated cleavage of 5′-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated <i>in vitro</i> when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the <i>in vitro</i> cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA <i>in vitro</i>.</p></div

PTPD reduces HBV replication in HBV infection models.

<p>(A) HepG2-hNTCP-C4 cells were pretreated with PTPD (5 μM) for 1 day, and subsequently infected with HBV in PTPD-containing culture medium. cccDNA levels at 3 dpi were analyzed by Southern blotting. (B) PXB cells (primary human hepatocytes) were pretreated with PTPD (5 μM) or 3TC (50 μM) from 1 day before infection to 7 dpi. Secreted HBV DNA levels were quantified at the indicated time points (left side). HBV RNA was quantified by RT-qPCR at 21 dpi (right side). Asterisks indicate statistically significant differences; *<i>P</i> < 0.05 compared with DMSO.</p

FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production.

<p>(A–D) Hep38.7-Tet cells were transfected with <i>FEN1</i>-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; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with siCtrl. (E–G) The pX330-FEN1 plasmid, which expresses Cas9 mRNA and sgRNA for exon 2 of the <i>FEN1</i> 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; *<i>P</i> < 0.05, **<i>P</i> < 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%.</p

The FEN1 inhibitor, PTPD, reduces cccDNA production.

<p>Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with DMSO (A–D). (E) RT-PCR analysis for pre-C mRNA transcribed from cccDNA in Hep38.7-Tet cells. β-actin was used as a loading control.</p

FEN1 protein facilitates cccDNA formation <i>in vitro</i>.

<p>(A) Schematic presentation of <i>in vitro</i> cccDNA formation assay. Purified NC-DNA (10⁸ copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; ***<i>P</i> < 0.001 compared with negative control (no enzyme). (C) Southern blot analysis of <i>in vitro</i> cccDNA formation assay. The DNA was analyzed directly (left), or treated with T5 exonuclease (middle) or T5 exonuclease, then subsequently digested with EcoRI (right). (D) Detection of RCA products. DNA treated with indicated enzymes was subjected to RCA and then digested with indicated restriction enzymes. Arrow indicates the 5.4-kb fragment of the HBV plasmid, used as a replication-defective control for transfection in E and F. (E and F) Equal amounts of digested RCA product generated in (D) were self-circularized and then transfected into HepG2 cells. Three days after transfection, HBV DNA was analyzed by qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences compared with the control; * <i>P</i> < 0.05, ** <i>P</i> < 0.01.</p

IgA switching activity correlates with reduction of HBV transcripts in B cells.

<p>(A, B, C, D) pPB and GFP expression vectors were transiently co-transfected into a mouse B cell line (CH12F3-2). Six hours after transfection, cells were divided into two groups and stimulated with (or without) CD40 ligand, IL-4, and TGF-β1 (CIT) for 3 days to induce IgA switching; (A) Schematic diagram of experimental design; (B) Nucleocapsid formation was measured using NAGE assays and GFP expression was used to confirm transfection. (C) HBV transcripts and AID expression levels were determined using qRT-PCR. (D) AID dependent IgA switching was determined using flow cytometry. (E, F, G, H) CH12F3-2 cells were co-transfected with pPB and the indicated siRNA against mouse AID (simAID-1 and -2) or controls (siCtrl and siGFP), and after 6 hours incubation, cells were further stimulated with CIT for 3 days. HBV transcript levels, knock down efficiency of AID, and IgA switching were determined using qRT-PCR, western blotting, and flow cytometry, respectively. (I, J, K) A tetracycline promoter-regulating HBV plasmid (pTre-HBV) was stably transfected into CH12F3-2 transfectants expressing tetracycline-responsible transactivator (Tet-off). Established CH12F3-2 transfectants were designated CH12-HBV; (I) Schematic diagram of CH12-HBV; (J) CH12-HBV cells were incubated in the presence or absence of CD40 ligand, IL-4, or TGF-β1 (CIT) and tetracycline as indicated for 2 days to induce endogenous AID expression and IgA switching. HBV transcription and AID expression levels were determined using qRT-PCR. (K) IgA switching was detected according to surface expression of IgA using flow cytometry. (L) AICDA-deficient and-wild type BL2 cells were transfected with HBV plasmid (pPB), and qRT-PCR was performed at 3 day post-transfection. *<i>P</i> < 0.05, **<i>P</i> < 0.01 (<i>t</i>-test). Data are representative of two to three independent experiments and error bars represent standard errors of the mean.</p

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

<div><p>Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner.</p></div

TGF-β1 upregulates APOBEC3 expression and suppresses HBV replication in Huh7 cells.

<p>Six hours after transfection of pPB, Huh7 cells were treated with TGF-β1 for 3 days, and HBV replication was evaluated. (A) qRT-PCR shows dose-dependent reduction of HBV transcripts by TGF-β1. (B) NC-DNA levels in secreted virions were also measured using qPCR. (C) Nucleocapsid NC-DNA and core protein levels in crude cytoplasmic extracts were assessed using NAGE assays. GAPDH protein levels in the same crude extracts were determined using western blotting. (D) Huh7 cells were treated with 150 ng/mL IL-4 or 10 ng/ml TGF-β1 for 3 days. Levels of HBV RNA and GAPDH mRNA were determined by Northern blot. Control: non-stimulated Huh7 cells. (E) Relative expression levels of APOBEC deaminases in non-stimulated Huh7 cells; Relative expression levels were determined using qPCR with cDNA from non-stimulated Huh7 cells and standard curves of control APOBEC deaminase DNA. Relative copy numbers of A3B were defined as one. (F) Induction of APOBEC deaminase expression in TGF-β1-treated Huh7 cells was estimated using qRT-PCR. Fold induction of APOBEC deaminases is shown in the top (10 ng/mL TGF-β1 for 24 or 48 h) and bottom (10 or 20 ng/mL TGF-β1 for 24 h) panels. (G) Huh7 cells were treated with indicated concentrations of TGF-β1 for 3 days. AID protein was immunoprecipitated using an anti-AID antibody (or an isotype control IgG, most right) and immunoprecipitated AID protein was determined by western blot. One lane contains immunoprecipitated protein harvested from 60% of 15 cm dish. All data are representative of two to four independent experiments. Error bars represent standard errors of the mean.</p

AID inducing HBV RNA reduction depends on Exosc3.

<p>(A, B) Huh7 cells were co-transfected with pPB and the indicated protein expression vectors, and were cultured for 3 days. Crude extracts (input) were then subjected to IP using an anti-FLAG antibody, and crude extracts and IP fractions were analyzed using western blotting. (C) Fold enrichment of HBV or HPRT transcripts upon anti-FLAG-Exosc3 IP; To determine RNA coprecipitation with the RNA exosome component Exosc3, Huh7 cells were transfected with pPB, pFLAG-Exosc3, and pCMV-AID (or pEGFP-C2), and were cultivated for 3 days. IP using anti-FLAG antibody was then performed, complexes of FLAG-Exosc3 were then eluted using free FLAG peptides, and the eluted RNA was analyzed using qRT-PCR. Combination of expression vectors used for transfection is the same with B (see numbers below the graph), and values in lane 3 were defined as 1. Error bars represent standard errors of the mean. (D) Associations of AID with RNA exosome proteins; Huh7 cells were co-transfected with indicated expression vectors, and were cultured for 3 days. Crude extracts (input) were subjected to IP with FLAG antibody, and crude extracts and IP fractions were analyzed using western blotting. Expression levels of GFP-Exosc7 were too low to be visualized in the crude extract (lanes 4 and 9, input), but GFP-Exosc7 was clearly detectable after FLAG-AID and FLAG-Exosc3 immunoprecipitation (lanes 4 and 9, IP). (E) Huh7 cells were co-transfected with pPB and either AID or GFP expression vectors and each of the siRNAs indicated in E and F, and cells were cultured for 3 days. HBV transcript levels, nucleocapsid formation, and Exosc3 expression were estimated using northern and western blotting, NAGE assays (E), and qRT-PCR analyses (F and G); siGFP and siCtrl were used as controls; **<i>P</i> < 0.01 (<i>t</i>-test); Data are representative of two to three independent experiments and error bars represent standard errors of the mean.</p

TGF-β1-mediated reduction of HBV transcripts depends on AID and Exosc3.

<p>Stable HBV transfectant Huh7 cells (7T7-8) were infected with recombinant lentiviruses to express indicated short hairpin (sh) RNA, and then cells were incubated in the presence or absence of 15 ng/ml TGF-β1 for 3 days. (A) Schematic diagram of experimental design; (B) AID expression levels in qRT-PCR and (C) IP western blotting. Crude extract before IP was also blotted (input). Crude extracts from TGF-β1-treated 7T7-8 transfectants were immunoprecipitated by anti-AID antibody. Loading control: anti-(adenosine deaminase acting on RNA) ADAR. (D) Exosc3 expression level in qRT-PCR or western blotting (E); shLuc was used as a control; (F) Reductions of HBV transcript levels following TGF-β1 treatment are compared between shAID-, shExosc3-, and shLuc-expressing 7T7-8 cells. HBV transcript levels of each non-stimulated transfectant are defined as 1; shLuc was used as a non-targeted control. *<i>P</i> < 0.05, **<i>P</i> < 0.01 (<i>t</i>-test), error bars represent standard errors of the mean. Data are representative of two to three independent experiments.</p