Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: 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 Huh7 NTCP 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 HepG2 NTCP 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.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Recombinant, Nucleic Acid Electrophoresis, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Amplification, Infection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: MC-HBV–based screen identifies cohesin complex as a novel host cccDNA binding factor. ( A ) MC-HBV was biotinylated and detected by dot blot using horseradish-peroxidase–streptavidin ( upper panel ); the input, unbound, and streptavidin-beads pull-down DNA was analyzed by dot blot to detect pull-down efficiency ( lower panel ). ( B ) Biotin-MC-HBV and MC-HBV were incubated with nuclear protein of HepG2 cells, and the interaction of H3, H3K27Ac, and H3K122me3 with cccDNA was identified by pull-down assay. ( C ) Schematic diagram of cccDNA-interacting factors screen with biotin-MC-HBV/pull-down/MS/bioinformatic analysis, biotin-linear-MC-HBV (linearized by EcoRI), and unlabeled MC-HBV taken as control. ( D ) Venn diagram of MS-identified proteins among 3 groups. A total of 306 candidates specifically were identified in the biotin-MC-HBV group and ( E ) analyzed for GO enrichment, or ( F, left panel ) were analyzed for enriched ontology clusters and a visualized network, ( F, right panel ) together with the 134 candidates classified into the DNA conformation regulation set. A circle node represented an enriched cluster, and nodes of the same color belong to the same cluster. ( G ) The protein–protein interaction network of these 134 proteins was mapped onto 6 pathways by MCODE ( left panel ), among which the chromosome segregation pathway was highlighted with red. Right : Cohesin composition and the information of 6 cohesin subunits and 2 regulators identified by MS. ( H ) A total of 1 μg biotin-MC-HBV, biotin-linear MC-HBV, and unlabeled MC-HBV were incubated with 400 μg HepG2 cells nuclear protein for 6 hours, then the interaction of SMC3 and SMC1A with cccDNA was identified by pull-down assay ( upper panel ); and 1 μg biotin-MC-HBV was incubated with 400 μg HepG2 cells nuclear protein with/without the presence of 1–3 μg unlabeled MC-HBV, then SMC3 were identified by pull-down assay ( lower panel ). ( I ) MC-HBV was transfected into Huh7 cells, or HBV-infected HLCZ01 cells and PHH cells at 200 genome equivalents (Geq), the enrichment of endogenous SMC3 and SMC1A on cccDNA was measured by ChIP assay (n = 3). ( J ) The green fluorescent protein (GFP)-SMC1A and GFP-SMC3 proteins were incubated with increasing concentrations of MC-HBV, the specific interactions were quantified by MST and plotted with the dissociation constant (Kd) equation, GFP protein served as control (n = 3). Data are presented as means ± SD. ∗∗ P < .01.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Binding Assay, Dot Blot, Incubation, Pull Down Assay, Control, Transfection, Infection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: Cohesin inhibits HBV replication. ( A ) Huh7 cells were transfected with Flag-SMC3 or siSMC3 for 72 hours, the expression of cohesin complex subunits was measured with Western blot. ( B ) Huh7 cells were transfected with MC-HBV for 3 days, while PHH, HepaRG NTCP , and HLCZ01 cells were infected with HBV at 200 genome equivalents (Geq) for 7 days. Then, cccDNA was extracted using a Hirt protein-free DNA extraction procedure and subjected to Southern blot assay. The cccDNA from the HepAD38 cell line was taken as a positive control. ( C and H ) In the MC-HBV/Huh7 model, MC-HBV was cotransfected with Flag-SMC3 or siSMC3 into Huh7 cells for 72 hours; in HBV-infected HLCZ01, PHH, and HepaRG NTCP models, cells were infected with HBV at 200 Geq for 24 hours and then transfected with Flag-SMC3 or siSMC3 for another 5 days, HBV replication was analyzed through measuring HBsAg/HBeAg (enzyme-linked immunosorbent assay), pgRNA, HBV DNA, and HBc antigen (HBcAg) (Western blot) (n = 3). ( I ) Huh7 cells were transfected with Flag-SMC3 and MC-HBV for 72 hours, and immunofluorescence staining was performed to detect Flag-SMC3 and HBsAg expression levels. Scale bar : 20 μm. ( J ) Huh7 cells were transfected with MC-HBV and siSMC1A or siRAD21 for 72 hours, and viral replication was analyzed (n = 3). Data are presented as means ± SD. ∗ P < .05, ∗∗ P < .01. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PF-DNA, protein-free DNA.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Transfection, Expressing, Western Blot, Infection, DNA Extraction, Southern Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: Cohesin reshapes cccDNA conformation to impede RNAPII recruitment. ( A ) In MC-HBV–transfected Huh7 and HBV-infected HLCZ01 models, SMC3 was overexpressed or knocked down, and cccDNA levels were detected by qPCR (n = 3). ( B ) Flag-SMC3 or siSMC3 was cotransfected with MC-HBV into Huh7 cells for 72 hours, enrichment of RNAPII and CTD-S2-RNAPII on cccDNA was measured by ChIP (n = 3). ( C ) Interaction of RNAPII with cohesin complex was analyzed by detecting SMC1/SMC3 enrichment using Co-IP assay. ( D ) Flag-SMC3 or siSMC3 was cotransfected with MC-HBV into Huh7 cells for 72 hours, and H3K27ac, H3K4me3, and H3K9me3 enrichment on cccDNA was measured by ChIP (n = 3). ( E ) Cohesin complex was purified from HepG2 cells with Co-IP using SMC1A antibody and glycine-HCl elution. ( F ) MC-HBV was incubated with purified cohesin complex at room temperature for 30 minutes, mock treatment and IgG precipitation were taken as control. MC-HBV conformation was analyzed with AFM imaging, and arrows indicate the classic cccDNA structure, the box represents the amplified region. Data are presented as means ± SD. ∗ P < .05, ∗∗ P < .01.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Transfection, Infection, Co-Immunoprecipitation Assay, Purification, Incubation, Control, Imaging, Amplification

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: Cohesin inhibits HBV transcription independently on SMC5/6 complex. ( A ) Huh7 cells were transfected with siSMC3 and MC-HBV for 72 hours, SMC3 enrichment on cccDNA was detected by ChIP (n = 3), RT-qPCR and Western blot measured SMC5 expression. ( B ) MC-HBV was cotransfected with Flag-SMC3 and siSMC5 into Huh7 cells for 72 hours, and HBsAg, HBeAg, and pgRNA levels were measured as described in Figure 3 A (n = 3). Data are presented as means ± SD. ∗ P < .05, ∗∗ P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: The loading of cohesin onto cccDNA is necessary for its antiviral role. ( A ) MC-HBV was cotransfected with siWAPL, siNIPBL, or siMAU2 into Huh7 cells for 72 hours, ( B ) HBV-infected (200 genome equivalents [Geq]) HLCZ01 cells for 24 hours, then siWAPL, siNIPBL, and siMAU2 were transfected for another 48 hours. RT-qPCR detected the knockdown effect and the enrichment of SMC3 on cccDNA was measured by ChIP (n = 3). ( C ) MC-HBV was cotransfected with Flag-SMC3 and siNIPBL or siMAU2 into Huh7 cells for 72 hours, and HBsAg, HBeAg, and pgRNA levels were measured (n = 3). ( D ) MC-HBV was cotransfected with WT SMC3 or SMC3-K38A mutant into Huh7 cells for 72 hours, and HBsAg, HBeAg, HBc, and pgRNA levels were measured (n = 3). SMC1A binding on cccDNA was detected by ChIP (n = 3). Data are presented as means ± SD. ∗ P < .05, ∗∗ P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Infection, Transfection, Quantitative RT-PCR, Knockdown, Mutagenesis, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: CTCF mediates cohesin loading onto HBV cccDNA. ( A ) HLCZ01 cells were infected with HBV (200 genome equivalents [Geq]) for 72 hours, the interaction of CTCF with cccDNA was analyzed by ChIP (n = 3). ( B ) The green fluorescent protein (GFP)-CTCF protein was incubated with increasing concentrations of MC-HBV, specific interactions were quantified by MST and plotted with the dissociation constant (Kd) equation, GFP protein served as control (n = 3). ( C ) Huh7 cells were transfected with Flag-CTCF and MC-HBV for 72 hours, binding of CTCF with cccDNA was first estimated with ChIP using anti-Flag antibody, after competitively eluting with Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DDDDK) peptide, reChIP assay was performed using an anti-SMC3 antibody to detect the occupancy of SMC3 with CTCF on cccDNA (n = 3). ( D ) siCTCF was cotransfected into Huh7 with MC-HBV for 72 hours. ChIP assay was performed with an anti-SMC3 antibody to analyze the enrichment of SMC3 on cccDNA (n = 3). ( E ) Co-IP was performed with an anti-CTCF antibody or IgG to analyze the interaction of CTCF with SMC1A and SMC3. ( F ) siSMC3 was transfected into Huh7 for 72 hours, and Co-IP was performed with an anti-CTCF antibody to analyze the interaction of CTCF and SMC1A. Relative quantification was performed by analyzing the relative ratio of band density using ImageJ software (National Institutes of Health, Bethesda, MD) as follows: relative SMC1A level interacting with CTCF = band density ratio (immunoprecipitated SMC1A/immunoprecipitated CTCF)/band density ratio (input SMC1A/input CTCF), scramble group set as 1. Data are presented as means ± SD. ∗∗ P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Infection, Incubation, Control, Transfection, Binding Assay, Co-Immunoprecipitation Assay, Quantitative Proteomics, Software, Immunoprecipitation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: The CTCF-mediated cohesin recruitment on cccDNA is necessary for cohesin-initiated inhibition on HBV replication. ( A ) MC-HBV was cotransfected with Flag-SMC3 and siCTCF into Huh7 cells for 72 hours, and HBsAg, HBeAg, HBc, pgRNA, and HBV-DNA levels were measured (n = 3). ( B ) The localization of CTCF-BS in the HBV genome and their sequence of WT and synonymous mutants. ※The CTCF-BS consensus sequence. ( C ) MC-HBV and CTCF-BS mutants were transfected into Huh7 for 72 hours, and the enrichment of CTCF on cccDNA was analyzed by ChIP (n = 3). ( D ) Sequence alignment of CTCF-BS1 and BS3 with different genotypes of HBV strains using the Clustal W algorithm of DNAstar Megalign software (Madison, WI). The dot represents the identical nucleotide; the red box indicates CTCF-BS1 and BS3 sequences. ( E ) MC-HBV and CTCF-BS mutants were transfected into Huh7 for 72 hours. The cccDNA binding of SMC3 was analyzed by ChIP (n = 3). ( F ) Flag-SMC3 was cotransfected with WT MC-HBV and CTCF-BS mutants (mBS1, mBS3) into Huh7 cells for 72 hours, HBsAg/HBeAg expression and pgRNA transcription were measured (n = 3). ( G ) WT, CTCF-mBS1, and CTCF-mBS3 mutants of MC-HBV were transfected into Huh7 for 72 hours, HBsAg/HBeAg expression and pgRNA transcription were measured (n = 3). Data are presented as means ± SD. ∗ P < .05, ∗∗ P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Inhibition, Sequencing, Transfection, Software, Binding Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: HBx transcriptionally represses SMC3 expression. ( A ) HLCZ01 and Huh7 NTCP cells were infected with HBV at 200 genome equivalents (Geq) for 5 days, Huh7 cells were transfected with MC-HBV for 3 days, and the messenger RNA (mRNA) and protein abundance of SMC3 were detected by RT-qPCR and Western blot. ( B ) The correlation analysis of immunohistochemical (IHC) staining of SMC3 and HBc antigen (HBcAg) in consecutive sections of para-nontumor tissue section obtained from HCC patients. ( C ) Huh7 cells were transfected with HBV1.1, HBc, HBx, and pre-S2 expression plasmids for 72 hours, SMC3 mRNA level was measured with RT-qPCR (n = 3) ( left panel ), HA-HBx plasmid at a different dose was transfected into Huh7 cells for 72 hours, and SMC3 protein was detected with Western blot ( right panel ). ( D ) HBx construct was transfected into HepG2 cells for 48 hours, SMC3 mRNA and protein expression were detected (n = 3). ( E ) MC-HBV and MC-HBVΔHBx or HBV1.1 and HBV1.1ΔHBx were transfected into Huh7 cells for 72 hours, and SMC3 mRNA and protein expression were detected. ( F ) Flag-SMC3 or Flag-SMC6 was cotransfected with HBx and HA-Ub into HEK293 cells for 48 hours, and then treated with MG132 for 4 hours. Flag-SMC6 ( left ) and Flag-SMC3 ( right ) were immunoprecipitated and immunoblotted with anti-HA antibody to measure their ubiquitylation. ( G ) HBx was overexpressed in Huh7 for 24 hours and then treated with 20 μmol/L nitazoxanide (NTZ) for 36 hours, SMC3 and SMC5 protein expressions were detected. ( H ) HBV1.1 and HBx constructs or HBx construct at different doses were cotransfected with SMC3 promoter-reporter, or HBx were cotransfected with a series of truncated SMC3 promoter-reporter constructs into Huh7 cells for 48 hours, the dual-luciferase assay was performed to detect SMC3 promoter activity (n = 3). ( I ) MAFG-siRNA or SMC5-siRNA were transfected into Huh7 cells for 24 hours and then the cells were cotransfected with SMC3 promoter-reporter and HBx expressing plasmid for 48 hours. The knockdown efficiency was evaluated by RT-qPCR, and SMC3 promoter activity was detected by dual luciferase assay (n = 3). Data are presented as means ± SD. ∗ P < .05, ∗∗ P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Expressing, Infection, Transfection, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Immunohistochemistry, Plasmid Preparation, Construct, Immunoprecipitation, Luciferase, Activity Assay, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: cccDNA Surrogate MC-HBV–Based Screen Identifies Cohesin Complex as a Novel HBV Restriction Factor

doi: 10.1016/j.jcmgh.2022.08.002

Figure Lengend Snippet: HBx promotes viral evasion from cohesin's restriction role. ( A ) MC-HBV and MC-HBVΔHBx were transfected into Huh7 cells for 3 days, ( B ) HBV-infected HepG2 NTCP cells for 24 hours, and then transfected HA-HBx for another 3 days. ChIP assay was performed to measure the enrichment of SMC3 on cccDNA (n = 3). ( C ) HBx construct was cotransfected with MC-HBV or MC-HBVΔHBx into Huh7 cells for 3 days, and ChIP assay was performed to measure the enrichment of SMC3 on cccDNA (n = 3) while SMC3 expression was analyzed by Western blot. ( D ) SMC3 siRNA was transfected into Huh7 for 24 hours, then the cells were transfected with MC-HBV or MC-HBVΔHBx for another 72 hours. HBV replication was analyzed (n = 3) and SMC3 expression was detected by Western blot. Data are presented as means ± SD. ∗ P < .05, ∗∗ P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Huh7 cells (3 × 10 7 ) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation.

Techniques: Transfection, Infection, Construct, Expressing, Western Blot