Fig 2: NTZ inhibits the HBx–DDB1 interaction. (A) HEK293T cells were transfected with plasmid expressing Flag-HBx, and treated with NTZ (10 µM) or DMSO (as a control) for 24 hours. Inhibition of the HBx–DDB1 interaction was examined through IP using anti-FLAG antibody, followed by Western blot analysis. Five percent of the total cell lysate was used as “input.” Representative results from 3 independent experiments are shown. Summarized results (n = 3) of relative band intensity are shown below the panels. *P = 6.8 × 10–5 (t test). (B) Inhibitory effects of the 5 candidate compounds on the HBx–DDB1 interaction. HEK293T cells were transfected with a plasmid expressing Flag-HBx, and treated with the indicated compounds (10 µM) for 24 hours. Inhibition of the HBx–DDB1 interaction was examined by IP using anti-FLAG antibody, followed by Western blot analysis. Representative results of 3 independent experiments are shown. Summarized results of relative band intensity are shown below the panels (n = 3). *P = 1.2× 10–3; **P = 2.1 × 10–3; ***P = 1.5 × 10–4 (t test). (C) HepG2 cells were transfected with a plasmid expressing Flag-HBx, and treated with NTZ (10 or 20 µM) or DMSO (as a control) for 24 hours. IP and Western blotting were performed as described in panel A. Representative results from 3 independent experiments are shown. Summarized results of relative band intensities are shown below the panels (n = 3). NS, not significant; *P = 1.3 × 10–3; **P = 1.4 × 10–4 (t test).

Fig 3: Characterization of recombinant HBx proteins. (A, top) TR-FRET analysis of the coexpressed His-HBx:GST-DDB1 complex in the presence of recombinant untagged HBx, SV5-V, DDB1, and BSA. (Bottom) TR-FRET analysis of the coexpressed His-HBx:GST-DDB1 complex in the presence of the wild-type (WT) HBx H-box peptide and a mutant HBx R96E H-box peptide. (B, top) Western blot analysis of PHH lysates collected 72 h after transduction with lentiviruses expressing GFP, Myc-HBx, Myc-HBx R96E, or the Myc-HBx-DDB1 fusion. HBx and the HBx-DDB1 fusion were detected using an anti-Myc antibody. Data are representative of results from 3 independent experiments. (Bottom) PHH were transduced with the same lentiviruses and 1 day later were infected with HBV?X. Extracellular HBeAg levels at day 13 postinfection are expressed as a percentage of the value for the HBx control; the bar height indicates the mean of data from 3 independent experiments, and the error bars represent the SEM. Statistical significance relative to the GFP control was calculated by one-way ANOVA with Dunnett’s multiple-comparison correction. ***, P < 0.001; ns, not significant (P > 0.05).

Fig 4: HBx binds to the C-terminal of TRIM5? BBox domain. (A,B) PHH cells were infected with HBV and subjected to HBx or DDB1 pull down assay, the whole-cell lysates were immunoblotted with TRIM5?, DDB1, HBx, and GAPDH antibodies. (C) HepG2 cells were transfected with Flag-HBx and HA-Ub plasmids, as indicated, 8 h before the collection, and the cells were treated by MG132 (10 uM) and the whole-cell lysates were immunoblotted with HA and Flag antibodies. (D) HepG2 WT cells were transfected with GFP-HBx and HA-TRIM5? plasmids, as indicated, and the whole-cell lysates were immunoblotted with HA, GFP, and GAPDH antibodies. (E) HepG2 cells were co-transfected with BBox and HBx WT or the R96E expression plasmids, as indicated, 36 h later, the whole cell lysates were immunoblotted with Flag, GST, or Tubulin antibodies. (F,G) 293T cells were transfected with Flag-HBx plasmids or co-transfected with BBox peptides expression plasmids, as indicated, and subjected to Co-IP analysis. The cell lysates and precipitated samples were subjected to immunoblotting using the indicated antibodies.

Fig 5: RepID-CRL4 ubiquitinates BUB3 during mitosis.a RepID-CRL4 localizes with BUB3 at the kinetochore in prometaphase cells. Immunofluorescence analysis of HCT116 RepID WT and KO cells was carried out in methanol-fixed cells using antibodies directed against CRL4 components (RepID, CUL4A, CUL4B, or DDB1; red signals) and BUB3 (green signal). CRL4 components do not associate with chromatin in the RepID KO cells because RepID, which recruits CRL4 to chromatin, is absent. Scale bar: 10 µm. b RepID-dependent CRL4 recruitment to chromatin throughout the cell cycle. Mitotic HCT116 RepID WT or KO cells were collected after a nocodazole block, reseeded in drug-free medium, harvested at the indicated time points, and chromatin-bound proteins were analyzed by immunoblotting and densitometry. Histone H3 and a-Tubulin were used as a loading control. c, d BUB3 is degraded in mitosis in RepID-expressing cells but not in RepID-deficient cells. HCT116 RepID WT or KO cells were released from mitotic block for the indicated time periods with or without the proteasome inhibitor MG132. Cell lysates were subjected to immunoblot analysis and densitometry. e BUB3 is ubiquitinated during mitosis in RepID-expressing cells but not in RepID-deficient cells. HIS-tagged ubiquitin plasmids were transiently transfected into HCT116 RepID WT and KO cells. Cells were collected at the indicated times after release from a nocodazole block, and his-tagged proteins from the released cells were isolated on Ni-NTA beads followed by immunoblot analysis. f Knockdown of CUL4 prevented the ubiquitination of BUB3 in RepID-expressing cells. HIS-ubiquitin plasmid and siRNA-CUL4 (directed against both CUL4A and CUL4B) were co-transfected into HCT116 RepID WT cells, which were synchronized, released, lysed, isolated with Ni-NTA beads, and analyzed for the presence of ubiquitinated BUB3 by immunoblotting as in e.