FIG. 3.
Expression of HBx protein interferes with cellular UDS. (A) Repair of damaged DNA in HBx-expressing cells. HepG2 cells were cotransfected with the pSV plasmid including the gene indicated below each bar and a luciferase reporter to control for transfection efficiency. Neor, neomycin resistance gene; HBx, X gene from HBV subtype adw2; X-rev, X in the reverse orientation; Luc, luciferase reporter gene alone. The number of micrograms of pSV-X transfected is shown below each bar in the righthand graph. Control pSV2neo plasmid was used to standardize the total amount of DNA introduced into the cells to 6 μg per plate. Repair for cells transfected with pSV2neo was set to 100%, and the repair measured for other transfected cells was compared to that value (numbers above each bar). Values shown are the means of at least three independent experiments; the error bars represent the corresponding standard deviations. (B) HBx expression. Duplicate 60-mm-diameter plates of cells transfected with pSV2neo (lanes 1 and 2) or pSV-X (lanes 3 and 4) were harvested 48 h posttransfection, pooled, and immunoprecipitated with anti-p28 negative control (odd-numbered lanes) or anti-X (even-numbered lanes). Liver extracts (25%, wt/vol) from ATX transgenic mice (38) were diluted 150-fold and then similarly immunoprecipitated and used as controls (lanes 5 and 6). Recovered proteins were transferred to nitrocellulose membrane for detection with anti-X by Western blotting. Migration of the 17-kDa HBx protein is shown at the right (arrowhead). (C) Transactivation by HBx. Cells were cotransfected with 5 μg of pSV-X and 1 μg of luciferase reporter under the control of one of several regulatory elements, noted below each bar: MSV, murine sarcoma virus; SV40, SV40 early promoter; ETS, regulatory element for the ets oncogene. The activation of each luciferase construct in the absence of HBx was normalized to 1.0 (black bars), and the fold activation of that basal level in the presence of HBx was calculated. Error bars represent the standard errors of the means from triplicate samples.