N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

Figure 4 Inhibitory effect of the N-terminal and C-terminal cytosine deaminase domain on HBV production in the co-transfected cells. (A and B) Human hepatoma HepG2 and HuH7cells were transiently co-transfected with the pHBV1.3 and CMV-driven expression vector encoding A3G and its N-terminal or C-terminal cytosine deaminase domain or with the empty vector pXF3H and pCMV-LacZ using Lipofectamine 2000 reagents. The cells were harvested 3 d after transfection. HBV core-associated viral DNA was prepared from nuclease-treated cytoplasmic lysates. Viral replicative DNA intermediates were analyzed by Southern blotting using a Dig-labeled full-length HBV DNA probe. (C) HBsAg and HBeAg levels were determined in the media of co-transfected HepG2 cells by ELISA, normalized to the activity of co-tansfected β-galactosidase in the cell lysates. The mean ± SE of six independent experiments is shown (error bar indicates standard error). (D) The hybridized HBV DNA from the replicative intermediates was quantified by radiophosphorimaging, normalized to the amount of secreted HBsAg (normalized for co-transfected β-galactosidase activity) and calculated relative to the empty vector control (pXF3H). The mean ± SE of six independent experiments is shown. (E) Anti-HA monoclonal antibody for Western blot analysis of cytoplasmic extracts from HepG2 cells co-transfected with the indicated plasmids. Numbers at the end or top of the lines indicate the amount of transfected plasmid DNA in micrograms. pXF3H: empty vector; pA3G: APOBEC3G expression plasmids; pNA3G: N-terminal cytosine deaminase domain of APOBEC3G expression plasmids; pCA3G: C-terminal cytosine deaminase domain of APOBEC3G expression plasmids; pEA3G: N-terminal region of APOBEC3G which does not contain any cytosine deaminase domain plasmid; HBV: hepatitis B virus; RC: relaxed circular DNA; SS: single-stranded DNA; HBcAg: hepatitis B virus core antigen; HBsAg: hepatitis B virus surface antigen; HBeAg: hepatitis B virus e antigen.