Fig. 7

rt269L attenuates HBx degradation through ubiquitination of HBx. A HepG2 cells were cotransfected with pHBx-GFP and mock, rt269L (genotype A or C), or rt269I (genotype A or C)-containing plasmids. Forty-eight hours posttransfection, the cells were treated with 100 µg/ml cyclohexamide to inhibit de novo translation. Cells were lysed at the indicated timepoints and analyzed by western blotting with anti-HBx or anti-GAPDH antibody. B HepG2 cells were cotransfected with pHBx-FLAG in combination with mock, rt269L (genotypes A and C), and rt269I (genotypes A and C) containing plasmids. Cells were treated with 20 µM MG132 at 42 h posttransfection for 6 h. Total cell lysates were immunoprecipitated using an anti-FLAG antibody and analyzed by western blotting using an anti-Ubiquitin antibody. C Level of HBx-GFP expression of a HBV subgenome-containing plasmid cotransfected into HepG2 cells, as indicated by fluorescence and flow cytometry assays. D HepG2 cells were cotransfected with pHBx-FLAG in combination with mock, Pol-, LHB-, and Core ORF-containing plasmids. Total cell lysates were immunoprecipitated using an anti-FLAG antibody and analyzed by western blotting using an anti-Ubiquitin antibody. E Detection of HBx-GFP protein expression in mock, rt269L, or rt269L/Pol∆ cotransfected HepG2 cells, as shown in fluorescence and western blot assays. F HepG2 cells were cotransfected with pHBx-FLAG in combination with mock and Pol-deleted rt269L/I plasmids. Total cell lysates were immunoprecipitated using an anti-FLAG antibody and analyzed by western blotting using an anti-Ubiquitin antibody. G A cycloheximide chase experiment with rt269L- or rt269I-containing Pol subgenome-carrying plasmid-transfected cells