FIG. 5.

The T552C mutation is responsible for efficient virion secretion by 4B and can confer a high-secretion phenotype to 3.4. (A) Cartoon view of the constructs. Clones 3.4 and 4B differ at three positions within the 520-to-730 region: 552, 636, and 705. Open ovals, wild-type 3.4 sequences; black squares, mutated 4B sequences. Eight site-directed mutants were prepared and tested. (B) HBV 1.5-mer DNA (1.5 μg) was transfected into Huh7 cells grown in six-well plates with the Mirus reagents and harvested 5 days later. HBsAg and HBeAg from culture supernatant were measured after 40- and 33-fold dilutions, respectively. HBV DNA replication inside transfected cells and virion secretion into the culture medium were analyzed by Southern blotting. DS, double-stranded DNA; SS, single-stranded DNA; HBV, a mixture of the EcoRI-linearized HBV genome (3.2 kb) and EcoRI/RsrII double-cut HBV DNA (1.7 and 1.5 kb).