Journal: International Journal of Biological Sciences

Article Title: Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular DNA-binding protein E2F4

doi: 10.7150/ijbs.62106

Figure Lengend Snippet: The comparison of Id1 expression in HBV or non-HBV context, and Id1 knockdown promoted HBV replication. (A) The Id1 mRNA (top layer) and protein (bottom layer) levels in 293T, L02,HepG2, Huh7,SMMC-7721,HepG2.2.15 and HepAD38 cells were analyzed by qPCR and western blotting with GAPDH used as the control gene. * P < 0.05, ** P < 0.01 and *** P < 0.001. n = 5. (B) The Id1 mRNA (top layer) and protein (bottom layer) levels in HepG2, HepG2.2.15 and HepG2-HBV1.1, which was transiently transfected with plasmid HBV1.1, were detected by qPCR and western blotting with GAPDH used as the control gene. ** P < 0.01. n = 5. (C) 3.5×10 5 or 7×10 5 Cells/35mm dish were infected with 400, 800 and 1600 HBV particles /cell. Then the cells were lysed and extracted for HBV DNA, which followed with southern blotting (top panel). Id1 protein levels of each cell dish infected with different virus titer of HBV were detected by western blotting (bottom panel). (D) Four plamids respectively expressing HBc/p/s/x proteins and HBV1.1 were transfected into HepG2 cells to verify the effect of HBV on Id1. (E) The HBc and Id1 protein level in HepAD38 cells with tetracycline-inhibiting HBV transcription was detected by western blotting with GAPDH used as the control gene. (F) HepG2.2.15 cells transfected with shRNA plasmids targeting Id1 or shCont vector as control group were harvested 72h, and intracellular HBV DNA was extracted and precipitated by isopropanol after viral infection . HBV DNA was determined by southern blotting and qPCR. Southern blotting analysis: Maker 1.8kb and 3.2kb; RcDNA, HBV relax circle DNA; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA. The qPCR data were expressed as the number of HBV DNA copies per cell. HBc and Id1 protein was determined by Western blotting. Meanwhile, GAPDH was used as a loading control. Expression of HBeAg in the supernatant of cell culture media were detected using ELISA 3 days after transfection. * P < 0.05, ** P < 0.01. n = 5.

Article Snippet: Cells were blocked by goat serum and then incubated with Id1 and E2F4 antibodies at room temperature for 1 h. Mouse anti-Id1 monoclonal antibody (sc-133104) and mouse anti-E2F4 monoclonal antibody (sc-511) was obtained from Santa Cruz Biotechnology (USA).

Techniques: Comparison, Expressing, Knockdown, Western Blot, Control, Transfection, Plasmid Preparation, Infection, Southern Blot, Virus, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular DNA-binding protein E2F4

doi: 10.7150/ijbs.62106

Figure Lengend Snippet: Enhancing expression of Id1 inhibited HBV transcription and replication in vitro and in vivo . (A) Overexpressing Id1 inhibited HBV DNA intermediates extracted from intracellular nucleocapsid 3 days after HepG2.2.15 cells infected with Id1 adeno-associated virus (Id1Ad). HBV DNA subsequently were subjected to qPCR and Southern blotting. The lysates from cells infected with Id1Ad were analyzed by western blotting with anti-Id1 and anti-HBc antibodies. GAPDH expression was used as loading control. HBeAg ELISA were used to screen culture supernatants 3 days after transfection. ** P < 0.01. n = 5. (B) HBV covalently closed circle DNA (cccDNA) and pgRNA were subjected to PCR quantification using specific primers with CA2 and GAPDH as the reference gene, respectively. * P < 0.05 and ** P < 0.01. n = 5. (C) HepG2-NTCP cells were firstly infected with 800 HBV viral particles/cell for 24 hours, and followed by transfection with pcDNA3.1-Id1 plasmid or pcDNA3.1 vector or medication with ETV. HBV DNA in cytoplasm was measured by qPCR after culture for additional 96 hours. All the qPCR data are presented as the mean±SE of triplicate experiments. ** P < 0.01. n = 5. (D) Effect of Id1 overexpression on four HBV promoters. Different luciferase reporter vectors were co-transfected with Id1Ad or GFPAd into HepG2 cells. pRL-TK was co-transfected to normalise the transfection efficiency. The luciferase activity was measured at 48 h post-transfection. * P < 0.05. n = 5. (E) The mice were randomly allotted to two groups of 5 individuals per group. 7×10 9 GFU Id1Ad (n1-n5) or GFPAd (n6-n10) were dissolved in 0.3 ml 0.9% normal saline and injected through the tail vein into mice. The mice were sacrificed at 20 days after injection, and the liver tissue and serum were collected. Intracellular HBV DNA extracted from 10 mg liver tissue was analyzed by southern blotting (top panel). The expression of Id1 and HBc in liver tissue lysates was tested by Western blotting with β-actin as an internal control (bottom panel). (F) The relative level of HBeAg in serum samples of AAV/HBV-infected mice were subjected to ELISA kits, ** P <0.01. n = 5.

Article Snippet: Cells were blocked by goat serum and then incubated with Id1 and E2F4 antibodies at room temperature for 1 h. Mouse anti-Id1 monoclonal antibody (sc-133104) and mouse anti-E2F4 monoclonal antibody (sc-511) was obtained from Santa Cruz Biotechnology (USA).

Techniques: Expressing, In Vitro, In Vivo, Infection, Virus, Southern Blot, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Saline, Injection

Journal: International Journal of Biological Sciences

Article Title: Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular DNA-binding protein E2F4

doi: 10.7150/ijbs.62106

Figure Lengend Snippet: Regulation of HBV transcription was involved in E2F4. (A) Genes including P16, P21, p53, NFԟB-p65, NFԟB-p50, VPS4b, HNF1ɑ, HNF4ɑ, HNF6ɑ, SNAI1, ZEB2, PPARɑ, RXRɑ, CREB2, SOX7, and TR2, which have been reported to play important roles in transcription or replication of HBV, were screened. qPCR analysis of gene expression of various transcription factors related to HBV replication in HepG2.2.15 after being infected with Id1Ad. n = 3. (B) The qPCR analysis was subjected to screen gene expression of various HLH transcription factors possibly related to Id1 in HepG2 transfected with HBV1.1 plasmid. GAPDH expression was used as internal control. * P < 0.05 and ** P < 0.01. n = 5. (C) Effects of a series of HLH transcription factors (including E2F4, CLOCK, TCF3, E40, USF1, HIF1ɑ ) overexpression on HBV Cp promoters were screened. Luciferase reporter vectors pGL3-Cp were cotransfected with overexpression plasmid into HepG2 cells. The cells were lysed and luciferase activity was determined at 48h after transfection. Meanwhile, the plasmid pRL-TK was used for normalizing the transfection efficiency. ** P <0.01. n = 5. (D) The comparison of E2F4 protein level among HepG2, HepG2-HBV1.1, HepG2.2.15 and HepAD38 (without tetracycline). (E) The E2F4 protein level in HepAD38 cells with tetracycline-inhibiting HBV transcription was detected by western blotting with GAPDH used as the control gene. (F) The qPCR analysis was subjected to screen gene expression of various E2F transcription factor in HepAD38 cells with tetracycline-inhibiting HBV transcription. ** P < 0.01. n = 5.

Article Snippet: Cells were blocked by goat serum and then incubated with Id1 and E2F4 antibodies at room temperature for 1 h. Mouse anti-Id1 monoclonal antibody (sc-133104) and mouse anti-E2F4 monoclonal antibody (sc-511) was obtained from Santa Cruz Biotechnology (USA).

Techniques: Gene Expression, Infection, Transfection, Plasmid Preparation, Expressing, Control, Over Expression, Luciferase, Activity Assay, Comparison, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular DNA-binding protein E2F4

doi: 10.7150/ijbs.62106

Figure Lengend Snippet: Transcription factor E2F4 was involved in Id1-mediated inhibition of HBV replication. (A) The location of Id1 (red) and E2F4 (green) in HepG2.215 cells under a natural state were detected by immunofluorescence images. DAPI (blue) were used to stain cell nucleus. Scale bar, 10 μm. (B) Lysates extracted from HepG2 cells expressing GFP-tagged E2F4 and Cherry-tagged Id1 were immunoprecipitated with anti-GFP or anti-Cherry, and the immunocomplexes were determined by immunoblot with respective anti-Cherry or anti-GFP antibody. (C) The western blot analysis following GST pull-down assay were performed with anti-Id1, anti-His, anti-E2F4, and anti-GST. The data showed that even though E2F4 expression in E. coli was unstable and easily degradable, Id1 also could be captured by E2F4. (D) Cytoplasmic-enriched(C) and nuclear-enriched(N) components from HepG2 cells (transfected with pCDNA3.1, pCDNA3.1+HBV1.1 and pcDNA3.1-Id1-Flag+HBV1.1, respectively) treated with a nuclear protein extraction kit were analyzed by immunoblot. Expression of GAPDH and PCNA, as markers for cytoplasm and nucleus respectively, was assessed by their specific monoclonal antibodies. The specificity of RB, p130, E2F4 and HBc bands were confirmed by their the specific antigenic peptides except Id1 by flag-antibody. (E) Southern blotting of HBV DNA was subjected to HepG2.2.15 cells after transfection early with shRNA-1/2 targeting Id1 and then infection with E2F4Ad. Southern blotting of HBV DNA was subjected to HepG2.2.15 cells after transfection early with shE2F4-1/2 targeting E2F4 and then infection with Id1Ad. The scramble control shRNA (shCont) was used as control group.

Article Snippet: Cells were blocked by goat serum and then incubated with Id1 and E2F4 antibodies at room temperature for 1 h. Mouse anti-Id1 monoclonal antibody (sc-133104) and mouse anti-E2F4 monoclonal antibody (sc-511) was obtained from Santa Cruz Biotechnology (USA).

Techniques: Inhibition, Immunofluorescence, Staining, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Transfection, Protein Extraction, Bioprocessing, Southern Blot, shRNA, Infection, Control

Journal: International Journal of Biological Sciences

Article Title: Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular DNA-binding protein E2F4

doi: 10.7150/ijbs.62106

Figure Lengend Snippet: Mutated HBV Cp binding site of 1758-TTAAAGGTC-1766 abolished E2F4-induced promotion of HBV and Id1-induced inhibition of HBV replication. (A) ChIP assays were performed to confirm the interaction between E2F4 and HBV Cp. Lysates extracted from HepG2.2.15 were immunoprecipitated with anti-E2F4, and the immunocomplexes were subjected to PCR with specific primers. ** P < 0.01. n = 3. (B) His-tagged pET28a-E2F4 1-180 constructs were expressed in Escherichia coli BL21 (DE3) cells induced with 0.2 mM IPTG. Truncated E2F4 1-180 protein were purified using Ni-NTA affinity column followed by stain with Coomassie Brilliant Blue dye. Ladder, protein molecular-mass markers; Lane 1, cell lysate after ultrasonication; Lane 2, cell supernatant after ultrasonication; Lane 3, Ni-NTA affinity column flow through fraction; Lane 4, eluted target protein with 20 mM imidazole; Lane 5, eluted target protein with 500 mM imidazole and followed with hyperfiltration. According the grayscale analysis by Image J software, the purity of E2F4 1-180 in the total eluted protein was higher than 90%. (C) The binding activity of E2F4 1-180 protein to the HBV Cp double-stranded probes amplificated by PCR presented in a dose-dependent mode, which determined by EMSA according to the manufacturer's protocol. Specificity of the DNA-protein complex was confirmed by competition with a 10-fold molar excess of unlabeled double-stranded DNA. BSA was used as negative control. (D) pGL3-Cp△site1, pGL3-Cp△site2 and pGL3-Cp△site3 were the deletion mutants of pGL3-Cp, whose deletion sites were respective nt1638-1644, nt1758-1766 and nt1798-1805. (E) pGL3-Cp△site2 resulted in E2F4 losing the ability to activating HBV Cp, which suggested nt1758-1766 should be the potential bound sites for E2F4. * P < 0.05 and ** P < 0.01. n = 5. (F) Interaction of HBV Core promoter truncation with E2F4 1-88 protein by ITC binding analysis. (G) The three-dimensional model of homodimer E2F4-DNA complex developed through homology modelling. Cartoon and electrostatic potential surface representation of the overall structure of the complex. A saturated red color indicates Ø<-10 kiloteslas/e and a saturated blue indicates Ø>10 kiloteslas/e; T = 293 K. (H) The enlarged image of the key residues at interaction sites between E2F4 and DNA fragment. The binding site is strongly positive charged region, and the sticks represent the 32 positive residues to interaction with DNA, including Arg17, His18, Glu19, Lys20, Lys28, Arg53, Glu54, Lys55, Arg56, Arg57, Asn60, Asn63, Lys73, Lys74, Lys76, and Asn77 from Chain A and B of the E2F4 homodimer. (I) When compared with HBV1.3-WT plasmids, a evident decrease of HBV1.3-mut (containing mutated E2F4 binding site) Cp DNA fraction bound to E2F4 was proved by CHIP performed in lysates extracted from HepG2 cells transfected with HBV1.3-mut. * P < 0.05 and ** P < 0.01. n = 3. (J) The capacity of E2F4 to enhancing HBV DNA copies in HepG2 cells transducted with HBV was weakened when the binding sites occurred mutant. ** P <0.01 and ns means no significance. n = 5. (K) The HepG2 cells infected with Id1Ad were cotransfected with plasmids HBV1.3-WT or HBV1.3-mut. The negative effect of Id1 on HBV replication and transcription were also abolished by binding sites mutant of E2F4. ** P <0.01 and ns means no significance. n = 5.

Article Snippet: Cells were blocked by goat serum and then incubated with Id1 and E2F4 antibodies at room temperature for 1 h. Mouse anti-Id1 monoclonal antibody (sc-133104) and mouse anti-E2F4 monoclonal antibody (sc-511) was obtained from Santa Cruz Biotechnology (USA).

Techniques: Binding Assay, Inhibition, Immunoprecipitation, Construct, Purification, Affinity Column, Staining, Software, Activity Assay, Negative Control, Transfection, Mutagenesis, Infection

Journal: International Journal of Biological Sciences

Article Title: Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular DNA-binding protein E2F4

doi: 10.7150/ijbs.62106

Figure Lengend Snippet: The comparison of Id1 and E2F4 expression in HBV-related HCC and schematic representation of the anti-HBV role of Id1. (A) The protein expression of E2F4, Id1 and HBc in TT and ANTT from 25 cases HBV-related HCC patient were tested by western blotting with β-actin used as the control protein. The right bar graph is the corresponding statistical figure. *P <0.05, **P < 0.01. n=25. (B) The conservatism of nt1758-1766 site sequence among four common HBV genotypes was shown. (C) HBV DNA were detected Id1 and E2F4 showed pan-genotypic antagonistic and synergistic effects on HBV respectively. * P < 0.05 and ** P < 0.01. n = 5. (D) Schematic representation of E2F4-induced activation of HBV transcription and the anti-HBV role of Id1. NTCP, sodium taurocholate cotransporting polypeptide; rcDNA; relaxed circular DNA, cccDNA, covalently closed circular DNA; pgRNA, pregenomic RNA; ER, endoplasmic reticulum; Golgi, Golgi apparatus.

Article Snippet: Cells were blocked by goat serum and then incubated with Id1 and E2F4 antibodies at room temperature for 1 h. Mouse anti-Id1 monoclonal antibody (sc-133104) and mouse anti-E2F4 monoclonal antibody (sc-511) was obtained from Santa Cruz Biotechnology (USA).

Techniques: Comparison, Expressing, Western Blot, Control, Sequencing, Activation Assay