hbx  (BioVendor Instruments)

Journal: JHEP Reports

Article Title: JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication

doi: 10.1016/j.jhepr.2023.100849

Figure Lengend Snippet: Knockdown of JMJD2D attenuates HBV transcription and replication. (A, B) Knockdown of JMJD2A-2D was used to measure the production of different HBV antigens. Knockdown of JMJD2D but not of the other JMJD2 family members ( JMJD2A-2C ) attenuated the protein levels of HBeAg (A), HBsAg (A), and HBx (B). (C) JMJD2D expression was reduced in JMJD2D -knockdown HepG2 cells which were transfected with HBV 1.3-fold genomic plasmid (pHBV1.3). (D) Knockdown of JMJD2D dramatically reduced the ratios of pgRNA/cccDNA and total RNA/cccDNA. HBV cccDNA, pgRNA, and total RNA were quantified by RT-qPCR. (E) Knockdown of JMJD2D significantly decreased the levels of extracellular HBV DNA. (F) The production of HBsAg and HBeAg were significantly decreased in JMJD2D -knockdown cells. (G) JMJD2D expression was reduced in JMJD2D -knockdown HBV-infected Huh-7-NTCP cells. (H) Knockdown of JMJD2D dramatically reduced the ratios of pgRNA/cccDNA and total RNA/cccDNA in HBV-infected Huh-7-NTCP cells. HBV cccDNA, pgRNA, and total RNA were quantified by RT-qPCR. The ratios of pgRNA to cccDNA and total RNA to cccDNA were calculated. (I) Knockdown of JMJD2D significantly decreased the level of extracellular HBV DNA HBV-infected Huh-7-NTCP cells. (J) Knockdown of JMJD2D reduced the production of HBsAg and HBeAg in HBV-infected Huh-7-NTCP cells. (K) JMJD2D knockout dramatically reduced the ratio of HBV pgRNA to cccDNA in mice livers that injected HBV DNA (Left), the production of HBsAg (middle) and HBeAg (Right) in the serum of mice, as measured by real-time qPCR and ELISA, respectively. For (A, D, F) and (H–K), data are from at least three independent experiments, presented as the mean ± SD of multiple samples. Ordinary one-way ANOVA analysis for D, F, and H–J, unpaired two-tailed Student’s t test for K, ∗∗ p <0 .01, ∗∗∗ p <0 .001, ∗∗∗∗ p <0 .0001. cccDNA, covalently closed circular DNA; HBx, Hepatitis B virus X protein; pgRNA, pregenomic RNA.

Article Snippet: The interaction between JMJD2D and HBx proteins was validated by co-immunoprecipitation (Co-IP) using magnetic protein A/G beads (MedChemExpress) conjugated to JMJD2D (Abcam, ab93694) and HBx (BioVendor, RD981038100) antibodies.

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Infection, Knock-Out, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Virus

Journal: JHEP Reports

Article Title: JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication

doi: 10.1016/j.jhepr.2023.100849

Figure Lengend Snippet: JMJD2D is a novel HBx-interacting protein. (A, B) Exogenous HBx interacted with JMJD2D in 293T cells, as determined by the Co-IP assay. (C, D) Endogenous HBx interacted with JMJD2D in HepAD38 cells, as determined by the Co-IP assay. (E) GST pull-down analysis of the interaction of HBx with different JMJD2D domains shown in the schematic diagram of the JMJD2D protein structure. (F) Schematic diagram showing the construction of GFP-tagged HBx truncation protein and the Co-IP assays were employed to validate the interaction of JMJD2D with truncated proteins of HBx. Co-IP, co-immunoprecipitation assay; HBx, Hepatitis B virus X protein.

Article Snippet: The interaction between JMJD2D and HBx proteins was validated by co-immunoprecipitation (Co-IP) using magnetic protein A/G beads (MedChemExpress) conjugated to JMJD2D (Abcam, ab93694) and HBx (BioVendor, RD981038100) antibodies.

Techniques: Co-Immunoprecipitation Assay, Virus

Journal: JHEP Reports

Article Title: JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication

doi: 10.1016/j.jhepr.2023.100849

Figure Lengend Snippet: JMJD2D enhances HBx protein expression by modulating its stability. (A) Knockdown of JMJD2D significantly decreased HBx protein levels in HBx-expressing HepG2 cells, as measured by western blotting. Quantification of HBx band intensity and normalisation with β-Actin as the loading control. (B) Knockdown of JMJD2D was unable to downregulate the mRNA levels of HBx, as quantified by Real-time qPCR. (C, D) Ectopic expression of JMJD2D significantly upregulated the protein level of HBx in 293T cells but failed to upregulate the mRNA levels of HBx. (E) Knockdown of JMJD2D dramatically reduced the protein stability of HBx in HepG2 cells after incubation with CHX at the indicated times. (F) Overexpression of JMJD2D sustains the protein stability of HBx in 293T cells after CHX treatment for the indicated time. A representative experiment is shown. The intensity of HBx protein was analysed and normalised with β-Actin. Data are from four independent experiments and one representative experiment is shown. For A–F, data are shown as mean ± SD. Ordinary one-way ANOVA analysis for A, B, E, and F, unpaired two-tailed Student’s t test for C, D, n.s., not significant, ∗ p <0 .05, ∗∗ p <0 .01. CHX, cycloheximide; HBx, Hepatitis B virus X protein; qPCR, quantitative PCR.

Article Snippet: The interaction between JMJD2D and HBx proteins was validated by co-immunoprecipitation (Co-IP) using magnetic protein A/G beads (MedChemExpress) conjugated to JMJD2D (Abcam, ab93694) and HBx (BioVendor, RD981038100) antibodies.

Techniques: Expressing, Western Blot, Incubation, Over Expression, Two Tailed Test, Virus, Real-time Polymerase Chain Reaction

Journal: JHEP Reports

Article Title: JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication

doi: 10.1016/j.jhepr.2023.100849

Figure Lengend Snippet: JMJD2D inhibits TRIM14-modulated ubiquitin-proteasome degradation of HBx protein. (A) MG132, a proteasome inhibitor, prevented the reduction of HBx protein levels caused by JMJD2D knockdown. Cells were treated with MG132 (20 μM) for 6 h and then protein lysates were used for western blotting to examine HBx protein levels. (B) Overexpression of JMJD2D prevented endogenous ubiquitin binding to HBx protein in 293T cells, the polyubiquitin ladder was detected by Co-IP coupled western blotting analysis. (C) Overexpression of JMJD2D prevented ectopic ubiquitin binding to HBx protein in 293T cells, the polyubiquitin ladder was detected by Co-IP coupled western blotting analysis. (D) TRIM14 interacted with HBx (Left) and JMJD2D (Right) in Huh-7 cells by Co-IP coupled western blotting assay. (E) Overexpression of JMJD2D prevented TRIM14-mediated reduction of HBx protein in Huh-7 cells as measured by western blotting. (F) Overexpression of JMJD2D blocked TRIM14-mediated ubiquitylation of HBx protein in Huh-7 cells, as examined by IP-coupled western blotting assay. Data were obtained from at least three independent experiments. For (A), data are presented as the mean ± SD of multiple samples and subjected to ordinary one-way ANOVA analysis, ∗∗ p <0 .01, ∗∗∗ p <0 .001. Co-IP, co-immunoprecipitation assay; HBx, Hepatitis B virus X protein; IP, immunoprecipitation.

Article Snippet: The interaction between JMJD2D and HBx proteins was validated by co-immunoprecipitation (Co-IP) using magnetic protein A/G beads (MedChemExpress) conjugated to JMJD2D (Abcam, ab93694) and HBx (BioVendor, RD981038100) antibodies.

Techniques: Western Blot, Over Expression, Binding Assay, Co-Immunoprecipitation Assay, Virus, Immunoprecipitation

Journal: JHEP Reports

Article Title: JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication

doi: 10.1016/j.jhepr.2023.100849

Figure Lengend Snippet: JMJD2D and HBx cooperatively promote HBV cccDNA transcription. (A) The ratios of pgRNA/cccDNA in JMJD2D and HBx co-transfected HBV-infected Huh-7 cells was significantly higher than those in cells transfected with JMJD2D or HBx alone, as measured by Real-time qPCR assay. HBV-infected Huh-7 cells were transfected with JMJD2D and gradient concentrations of HBx plasmids and the ratio of pgRNA/cccDNA was calculated. (B) Schematic illustration of the construction of HBx-deleted HBV DNA plasmid. HBV-ΔX represents HBx deleted HBV. (C) Western blotting failed to detect HBx protein expression in Huh-7-shJMJD2D cells that were transfected with HBV-ΔX DNA. (D) HBV-ΔX-infected Huh-7-shJMJD2D cells exhibited the lowest levels of viral antigen compared with HBV-infected Huh-7-shCTL cells, HBV-infected Huh-7-shJMJD2D cells, and HBV-ΔX-infected Huh-7-shCTL cells, as measured by ELISA assay. (E) The recruitment of JMJD2D and HBx on HBV cccDNA was detected by ChIP assay in HBV-infected Huh-7-NTCP cells. (F) HBx deficiency remarkably reduced JMJD2D recruitment of HBV cccDNA, as examined by ChIP-qPCR assay. (G) The enrichment of H3K9me3 on HBV cccDNA was significantly increased after knockdown of JMJD2D , HBx absence, or both, as measured by ChIP-qPCR using H3K9me3 antibodies. Representative data are from at least three independent experiments. Data are presented as the mean ± SD of multiple samples. Unpaired two-tailed Student’s t test for E, and ordinary one-way ANOVA analysis for A, D, F–H and J, ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, n.s., not significant. ChIP, chromatin immunoprecipitation; cccDNA, covalently closed circular DNA; HBx, Hepatitis B virus X protein.

Article Snippet: The interaction between JMJD2D and HBx proteins was validated by co-immunoprecipitation (Co-IP) using magnetic protein A/G beads (MedChemExpress) conjugated to JMJD2D (Abcam, ab93694) and HBx (BioVendor, RD981038100) antibodies.

Techniques: Transfection, Infection, Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Chromatin Immunoprecipitation, Virus

Journal: JHEP Reports

Article Title: JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication

doi: 10.1016/j.jhepr.2023.100849

Figure Lengend Snippet: JMJD2D-specific inhibitor 5C-8-HQ effectively interferes with HBV transcription and replication. (A) The protein levels of HBx were reduced in HBV-infected HepG2-NTCP cells after 5C-8-HQ (20 μM) and IFNα (20 ng/ml) treatment, respectively, as measured by western blotting. (B) The ratios of pgRNA/cccDNA and HBV total RNA/cccDNA were significantly decreased in HBV-infected HepG2 cells after 5C-8-HQ and IFNα treatment, respectively. (C) Schematic illustration of the experimental strategy of HBV-infected differentiated HepaRG cells (Left). The ratios of pgRNA/cccDNA and HBV total RNA/cccDNA were also significantly decreased in HBV-infected differentiated HepaRG cells (Right). (D) Schematic overview of the experimental strategy: C57BL/6 mice were hydrodynamically injected with HBV DNA and treated with 5C-8-HQ or IFNα. (E) HBV pgRNA levels were reduced in HBV-injected mice after 5C-8-HQ and IFNα treatment, respectively, as analysed by qPCR (n = 7). (F) Levels of HBV antigens including HBeAg (Left) and HBsAg (Right) in the mouse serum were decreased after 5C-8-HQ and IFNα treatment, respectively, as measured by ELISA (n = 8). Data are presented as the mean ± SD of multiple samples. Ordinary one-way ANOVA analysis for B, C, unpaired two-tailed Student’s t test for E, F, ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001. (G) Schematic presentation of the mechanisms by which JMJD2D promotes HBV transcription and replication. HBV infection activates phosphorylation of p65 in NF-κB signalling to induce JMJD2D expression, then JMJD2D stabilises HBx protein by preventing TRIM14-mediated ubiquitin-proteasome degradation of HBx and cooperates with HBx to promote cccDNA transcription by demethylating H3K9me3 on cccDNA and preventing p53 binding to cccDNA. cccDNA, covalently closed circular DNA; HBx, Hepatitis B virus X protein; pgRNA, pregenomic RNA.

Article Snippet: The interaction between JMJD2D and HBx proteins was validated by co-immunoprecipitation (Co-IP) using magnetic protein A/G beads (MedChemExpress) conjugated to JMJD2D (Abcam, ab93694) and HBx (BioVendor, RD981038100) antibodies.

Techniques: Infection, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Expressing, Binding Assay, Virus

Journal: Journal of Virology

Article Title: Peroxiredoxin 1, a Novel HBx-Interacting Protein, Interacts with Exosome Component 5 and Negatively Regulates Hepatitis B Virus (HBV) Propagation through Degradation of HBV RNA

doi: 10.1128/JVI.02203-18

Figure Lengend Snippet: HBx and Prdx1 were predominantly colocalized in the cytoplasm. Huh-7.5 cells were transfected with expression plasmids for HBx-Myc-His6 (top panel), HBx(C17A)-Myc-His6 (second panel), FLAG-Prdx1 (third panel), HBx-Myc-His6 and FLAG-Prdx1 (fourth panel), or HBx(C17A)-Myc-His6 and FLAG-Prdx1 (bottom panel), respectively. At 48 h posttransfection, the cells were stained with anti-c-Myc mouse monoclonal antibody, followed by Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and anti-FLAG rabbit polyclonal antibody, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). The cells were stained with Hoechst 33342 for the nuclei (blue). These stained cells were examined using a Zeiss LSM700 scanning laser confocal microscope. Scale bar, 20 μm.

Article Snippet: The primary antibodies used in this study were mouse monoclonal antibodies against c-Myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), FLAG (Clone M2; Sigma), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, Billerica, MA); rabbit monoclonal antibody against Prdx1 (Cell Signaling Technology, Danvers, MA); rabbit polyclonal antibody against HBx (Abcam, Cambridge, UK), HBc ( 62 ), and Exosc5 (Novusbio, Littleton, CO); and horse polyclonal antibody against HBs (Abcam).

Techniques: Transfection, Expressing, Staining, Microscopy