FIGURE 4.

HBV expression is not affected by enhanced or reduced expression of ARE-regulated genes. HepAD38 were cotransfected with expression vectors encoding YFP-NQO1 or YFP-γ-GCS-fusion proteins. Cotransfection with YFP served as control. a, secretion of HBsAg and of HBeAg was quantified by ELISA. b, confocal laser scanning immunofluorescence microscopy (630-fold magnification). LHBs was detected by MA18/7 (red fluorescence) and YFP-NQO1 or YFP-γ-GCS (green fluorescence); nuclei were stained with DAPI. c, Western blot analysis of cellular lysates using an LHB-specific antiserum (MA18/7). Expression of the YFP-NQO1 or YFP-γ-GCS fusion proteins was demonstrated by NQO1- or γ-GCS-specific antibodies. d and e, HepAD38 cells were cotransfected with pcaNrf2 or ptdnNrf2. Transfection with pCDNA.3 served as control. The amount of secreted HBeAg (d) was determined by ELISA. The amount of secreted viral particles was quantified by real time PCR (e). f, primary mouse hepatocytes isolated from Nrf2^−/− mice or from the corresponding WT mice were infected with AdHBV. The expression of HBV was quantified by HBsAg- and HBeAg-specific ELISA.