FIG. 8.

Effects of core promoter and precore mutations on viral replication and protein expression. Replication-competent constructs containing either wild-type adw (WTadw), precore (WTadwpreC⁻ or MT5/6preC⁻, each containing a mutated precore start codon), or core promoter mutant sequences (Table 1) were transfected into HuH-7 cells. The transfected cells were analyzed for viral replication, core protein expression, and HBeAg and HBsAg synthesis. (A) Viral replication. Viral replicative intermediates were analyzed by Southern blotting of core particle-associated viral DNA. SS, single stranded DNA. (B) Core protein expression. Transfected cell lysates from the experiment shown in panel A were subjected to SDS-PAGE and immunoblotting with an anticore antibody. Positions of molecular weight (MW) markers (in kilodaltons) are indicated on the left. (C) HBeAg and HBsAg synthesis. HBeAg (left) and HBsAg (right) were analyzed from the medium of transfected HuH-7 cells by using radioimmunoassays. Transfection efficiencies as monitored by pTKGH cotransfection were similar among all samples. Results are shown as signal-to-noise ratio (S/N) of three independent experiments (average ± standard deviation).