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: Rabbit anti-HBc poly-clonal antibody (B0586) was obtained from Dako (Denmark).
Techniques: Expressing, In Vitro, In Vivo, Infection, Southern Blot, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Injection
