Fig. 2

Infection with PreS variants that altered AA properties led to envelop protein retention within hepatocytes. A Schematic PreS/S regions of Ref-HBV, mtPreS1 and mtPreS2 based on the isolates obtained from 35 HBsAg-seronegative HCC patients. Shown are the predicted 3D structures of large envelop proteins encoded by Ref-HBV, mtPreS1 and mtPreS2 respectively using the RaptorX structure prediction sever (http://raptorx.uchicago.edu/). B, C In 8-well chamber slides, 8 × 10⁴/well of HepG2 cells were transfected with 300 ng of Ref-HBV, mtPreS1 or mtPreS2 plasmids respectively. Forty-eight hours later, the cells were double-stained B with antibodies against HBsAg (green) and calnexin (red, ER marker) or double-stained C against HBsAg and mitochondria (red) with Mito-Tracker Red CMXRos. Representative images are one of three independent experiments. DAPI (blue)-stained nuclei. D In 6-well plates, 5 × 10⁵/well of HepG2 cells were transfected with 2.5 μg of different HBV plasmids, respectively, and cultured for 48 h. The supernatant was collected, and cells were washed twice with PBS and harvested. Hepatocyte ER was further separated by density gradient ultracentrifugation from total cellular proteins. HBsAg in supernatant (secreted) and in total cell lysates (intracellular) were quantified using an ELISA kit. Bar graphs (mean ± SEM) show the ratio of intracellular to secreted HBsAg after normalizing to total cellular proteins (left), and the amount of HBsAg in ER organelle (right), from three independent experiments. Each group was triplicated in one experiment. The same amount of pcDNA3.1 (Vec) was used as transfection control. The map of different plasmids was provided in Additional file 1: Fig. S3. Differences of the HBsAg secretion in differently transfected cells were compared with one-way ANOVA analysis. *P < 0.05, **P < 0.01, ***P < 0.001 between Ref-HBV and mtPreS1 or mtPreS2; ^#P < 0.05, ^##P < 0.01 between mtPreS1 and mtPreS2