CRISPR-mediated proximity labeling unveils ABHD14B as a host factor to regulate HBV cccDNA transcriptional activity

Abstract

Background: The long-term goal of chronic hepatitis B research is a functional cure (HBsAg seroclearance). Although currently used nucleos(t)ide analogs can efficiently inhibit viral replication, they do not reduce viral RNAs or proteins produced from covalently closed circular DNA (cccDNA), and rarely achieve a functional cure. To overcome this situation, revealing the mode of the existence of cccDNA is required, including identifying the interreacting proteins with cccDNA. Here, we aimed to identify novel proteins that interact with cccDNA.

Methods: Using an in vitro HBV infection model and a sequence-specific proximity labelling method consisting of dead Cas9 and biotin identification (BioID2), we comprehensively determined proteins that possibly interact with cccDNA. After identifying the candidate proteins, the HBV RNA transcription levels were examined by knocking out the associated genes.

Results: We identified ABHD14B as a protein that interacts with cccDNA and inhibits HBV RNA transcription from cccDNA. ABHD14B decreases the acetylation levels of histone proteins that control the transcription levels of HBV RNA in cccDNA. Moreover, ABHD14B interacts with TFII-I, which binds directly to cccDNA in a sequence-dependent manner. These results suggest that the host protein, ABHD14B, is recruited to cccDNA via the TFII-I protein, inhibiting HBV RNA transcription from cccDNA by deacetylating cccDNA histones.

Conclusions: ABHD14B was newly identified as a suppressor of HBV RNA transcription from cccDNA, which may improve our understanding of the mode of existence of cccDNA, providing a basis for development of a functional cure.

Keywords: ABHD14B; HBV; cccDNA; dCas9; proximity labelling.

Graphical abstract

FIGURE 1

Establishment of a screening system. (A) Overview of the screening strategy. (B, C) The establishment of NTCP and dCas9-BioID2 co-expressing HepG2 cells (HepG2-NTCP; dCas9-BioID2) was confirmed using western blotting (B) and flow cytometry (C). (D) Time course of experiments using HepG2-NTCP and dCas9-BioID2 cells. (E–H) The HBV infection ability of HepG2-NTCP dCas9-BioID2 cells was determined using western blotting (E), ELISA (F), and qPCR (G). cccDNA levels were determined on day 7 after infection using the droplet digital PCR (H). (I) Histone H3 protein was detected using this system. Representative images from 3 independent experiments are shown. Abbreviations: BioID, biotin identification; COI, Cutoff Index; dCas9, dead Cas9; M.C., media change; NTCP, sodium taurocholate co-transporting polypeptide; sgRNA, single-guide RNA.

FIGURE 2

Identification of host proteins that interact with HBV cccDNA minichromosome. (A) Protein detection using silver staining. Bands corresponding to molecular weights of 27, 22, and 15 kDa (indicated by red arrows) were excised and analyzed using mass spectrometry. (B) Proteins identified using LC-MS/MS were verified by western blotting. (C) ChIP assay confirming the interaction between HBV cccDNA and the identified proteins. Data are means±SDs (n=6). Abbreviations: BioID, biotin identification; cccDNA, covalently closed circular DNA; ChIP, chromatin immunoprecipitation; dCas9, dead Cas9; NTCP, sodium taurocholate co-transporting polypeptide; sgRNA, single-guide RNA.

FIGURE 3

ABHD14B regulates viral transcription from HBV cccDNA. (A) Strategy to create a customized CRISPR knockout cell mini library for functional screening. (B) Time course of the experiment using the CRISPR knockout cell mini library. (C) Transcriptional activity of HBV in each knockout cell line was determined using RT-qPCR. Data are means±SDs (n=3). *p=2.3×10^–3; ** p=0.018. Statistical analyses were performed using Welch t test. (D) Viral surface antigen levels were determined using western blotting. Representative images from 3 independent experiments are shown. The summarized results for the relative band intensities (n=3) are shown below the panels. *p=6.0×10^–3. Statistical analyses were performed using Welch t test. (E) HBe Ag levels in culture supernatants were determined using ELISA. *p=8.2×10^–5; ** p=4.8×10^–4. KO-GFP; cells with CRISPR construct targeting GFP, as a control. KO-ABHD14B; cells with the CRISPR construct targeting ABHD14B. Statistical analyses were performed using Welch t test. (F) HBV attachment and internalization activities were determined using RT-qPCR (middle, attachment, right, and internalization). Statistical analyses were performed using Welch t test. ΔGFP; cells with CRISPR construct targeting GFP, as a control. The levels from ΔGFP cells were set as 1.0. (G) HBV transcriptional activity was determined using a Gaussia luciferase (Gluc) reporter assay with a minicircle HBV model. *p=9.5×10^–3. Statistical analyses were performed using Welch t test. Abbreviations: cccDNA, covalently closed circular DNA; COI; Cutoff Index.​​​​​; KO-GFP, xxx; M.C., media change​​​​​​; NTCP, sodium taurocholate cotransporting polypeptide; sgRNA, single-guide RNA.

FIGURE 4

ABHD14B reduces histone acetylation on HBV cccDNA minichromosome. (A) The status of histones (acetylation of H3K27 and methylation of H3K9) on HBV cccDNA (left) and on RPL30 as a control (right) was determined using a ChIP assay with NTCP-HepG2 and NTCP-HepG2 with ABHD14B knockout cells. The ChIP assay was performed 6 days after HBV infection. Data are means±SDs (n=3). Statistical analyses were performed using Welch t test. (B, C) ABHD14B does not interact with HBV core or HBx proteins. HepG2 cells were transiently transfected with HA-ABHD14B expressing plasmid together with a flag-HBV core or flag-HBx protein-expressing plasmid, followed by immunoprecipitation using an anti-flag. Representative images from 3 independent experiments are shown. (D) TFII-I was identified as the binding protein to ABHD14B. ABHD14B protein was immunoprecipitated with anti-HA using HA-ABHD14B stably expressing NTCP-HepG2 cells. Mass spectrometry identified TFII-I as the binding partner of ABHD14B. Peptide sequences with yellow markings were identified by mass spectrometry. (E) Endogenous TFII-I was immunoblotted using immunoprecipitated samples. Representative images from 3 independent experiments are shown. Aliquots of each sample before IP (5%) were used as input. ns and nonspecific bands. (F) consensus sequence (BRGATTRBR) for TFII-I-binding sites in the HBV genome. (G) TFII-I interactions with HBV sequences determined by ChIP analysis. Three days after transfection with the minicircle HBV construct in HepG2 cells, ChIP analyses were performed using an anti-TFII-I antibody. PCR was performed with primer pairs amplifying the region containing the TFII-I consensus sequences and with primer pairs amplifying unrelated regions in the HBV genome. Data are means±SDs (n=3). Statistical analyses were performed using Welch t test. Abbreviations: cccDNA, covalently closed circular DNA; ChIP, chromatin immunoprecipitation; NTCP, sodium taurocholate co-transporting polypeptide.

FIGURE 5

TFII-I is crucial for the deacetylation of histone on HBV cccDNA. (A) TFII-I was knocked out using CRISPR-mediated mutagenesis in NTCP-HepG2 cells. (B, C) HBV transcription levels in HBV cccDNA were reduced in TFII-I-knockout cells. The minicircle HBV luciferase reporter or HBV was transfected into NTCP-HepG2 cells. Five days after transfection or infection, luciferase values (B) and HBV RNA levels (C) were determined. Data are means±SDs (n=3). Statistical analyses were performed using Welch t test. (D) TFII-I is involved in the deacetylation of H3K27 on HBV cccDNA, as determined by ChIP assay. Three days after transfection of the mini-circular HBV construct into NTCP-HepG2 cells or NTCP-HepG2 cells with TFII-I knockout, a ChIP assay was performed using an antiacetylated H3K27 antibody. Data are means±SDs (n=3). Statistical analyses were performed using Welch t test. (E) TFII-I is involved in the deacetylation of H3K27 in HBV cccDNA in a natural HBV infection model. Six days after HBV infection of NTCP-HepG2 cells or NTCP-HepG2 cells with TFII-I knockout, ChIP assays were performed using antiacetylated H3K27 or anti-TFII-I antibodies. Data are means±SDs (n=3). Statistical analyses were performed using Welch t test. Abbreviations: cccDNA, covalently closed circular DNA; NTCP, sodium taurocholate co-transporting polypeptide.