Figure 4. ZAP-mediated HBV RNA reduction primarily occurs in the nucleus.

(A) Intracellular localization of ZAP-S by microscopic immunofluorescence analysis. HepG2 cells were transfected with HA-tagged ZAP-S expression plasmid. Cellular distribution of ZAP-S was stained with HA antibodies and corresponding fluorescence-labeled secondary antibodies (left panel). DAPI staining of nucleus is shown in the middle panel. Merged signals of ZAP-S and nucleus are shown in the right panel. (B) Subcellular distribution and antiviral activity of ZAP-S. HepG2 cells in 35 mm dishes were transfected with 2 µg of plasmid pHBV1.3 and 2 µg of ZAP-S expression vector. Cells were harvested at day 4 post transfection. Cell fractionations for RNA and protein analysis were performed as described in the Materials and Methods. Total cellular RNA, cytoplasmic and nuclear RNA were isolated and subjected to Northern blot analysis of HBV RNA (upper panel). Subcellular distribution of ZAP-S was revealed by Western blot using HA antibodies, with β-actin serving as loading control (lowers panels). Annexin and Lamin A/C Western blots were used to confirm the purity of cytoplasmic and nuclear fraction, respectively (panel C).