Identification and characterization of a novel bipartite nuclear localization signal in the hepatitis B virus polymerase

Figure 2 Hepatitis B virus polymerase harbors a functional nuclear localization signal in the terminal protein domain. A-D: HuH-7 cells were transfected with (A) the negative control wild type green fluorescent protein (GFP) (pEGFP-N1), (B) a positive control: GFP fused to a prototype bipartite nuclear localization signal (NLS) of human nucleoplasmin, (C) GFP fused to the putative bipartite NLS of hepatitis B virus (HBV) P protein, (D) the same as (C) but cells were treated with 10 × IC₅₀ of casein kinase II (CKII) inhibitor 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) 2 h prior analysis. The fluorescence was measured in living cells by confocal laser scan analysis. The central layer (out of 6) was quantitated along the red indicated line and displayed in the corresponding graph of the lower panel as relative fluorescence intensity [I]. Differences of mean fluorescence intensities in the cytoplasm and within the nucleus (indicated as black line in the graph) were calculated and are indicated in percent. One representative cell for each fusion protein is shown; E: Confocal immunofluorescence microscopy of HuH-7 cells transfected with an expression vector encoding wt P or the mutant ∆CKII (= T100I) that is not phosphorylated by CKII. For detection of P (red) a rabbit-derived spacer domain-specific serum was used. Nuclei were stained with DAPI (blue); F: HuH-7 cells were transfect with the indicated expression vectors and left untreated or treated with 10× IC₅₀ of CKII inhibitor DMAT 5 h prior analysis. Transfection with pCDNA.3 served as control. Cells were lysed, the cytosolic and nuclear fraction were isolated by differential centrifugation and analyzed by western blotting. For detection of P a TP-domain specific serum was used. Detection of hexokinase and of lamin served as loading control and as control for the purity of the subcellular fractions. All experiments were performed in triplicate. One representative is shown.