Fig. 4

Secretion of genome-containing virions is also affected by proline substitution in the small loop at amino acid 196–198 in a position-dependent manner. A HuH-7 cells was cotransfected with a polymerase-deficient replicon plasmid and a wild type polymerase expression vector (pMT-pol). Surprisingly, unlike mutants 196P and 198P, a proline substitution at amino acid 197 resulted in no virion secretion deficiency. Secreted viral particles in the media were enriched by PEG before native agarose gel, followed by Western and Southern blot analyses. Lane 1: A very faint and weak HBV DNA signal can still be visualized from the Pol-null replicon in both intracellular and extracellular panels. Under the strong CMV promoter in the context of pCHT-9/3091 (Materials and Methods), this Pol-null replicon is a bit leaky in polymerase expression and viral DNA synthesis. The virion-associated DNAs were quantified by densitometry and Image J software. B Virion-associated HBV DNA genome in the supernatant of HuH-7 cells was analyzed by qPCR. HBV virions were immunoprecipitated with a rabbit anti-HBs antibody and treated with nuclease digestion before viral DNA extraction. HBV DNA signals of small loop mutants cotransfected with pMT-Pol were compared individually to the WT-HBV control. GE: genomic equivalent. Bar graph statistics by one-way ANOVA tests. ***P < 0.001; *P < 0.05