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

Figure 4 Casein kinase II inhibition impairs hepatitis B virus replication in infected primary Tupaia hepatocytes. A: HuH-7 cells were transfected with mutant versions of a 1.2 fold hepatitis B virus (HBV) genome. After 5 d the secreted viral particles were precipitated with a HBs specific antibody and the containing 3.2 kb HBV genomes were visualized by the radioactive tracer [α-³²P] dCTP incorporated using the endogenous polymerase activity. The unphosphorylated form of the casein kinase II (CKII) recognition site in the P protein was simulated by a T100I substitution (∆) and the pseudo-phosphorylation was simulated by a T100D substitution (*) in the 1.2 fold HBV wild type genome. The nuclear localization signal (NLS) was inactivated by mutating the downstream basic cluster (K105D and K106S) on the P protein. A 2.5 × HBV genome and a P deficient genome [HBV (P-)] served as controls; B: Primary Tupaia hepatocytes were infected with HepAD38 derived HBV and post infection treated for 36 h with solvent DMSO or 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) (10 × IC₅₀). Twelve days after infection the HBV genome secretion was measured by Lightcycler polymerase chain reaction. The cccDNA content of infected Tupaia hepatocytes that were incubated for 36 h with the indicated amounts of DMAT was visualized by Southern blot using a HBV specific probe. The specificity of DMAT incubation was analyzed by 2 h inhibitor pre-treatment of the stably HBV transfected cell line HepG2.2.15 followed by treatment of 36 h with 10 × IC₅₀ CKII inhibitor DMAT (IC₅₀ in rat liver = 150 nmol/L) and genome secretion was compared to the solvent control DMSO measured by Lightcycler PCR. All experiments were performed in triplicate. The figure shows one representative experiment; C, D: Primary Tupaia hepatocytes were infected with HepAD38-derived HBV and at day 1, day 2, day 4 and day 7 treated for 36 h DMAT (10 × IC₅₀). Twelve days after infection the HBV replication was measured by HBeAg or HBsAg-specific enzyme linked immunosorbent assay. The bars represent the standard deviation.