Hepatitis B doubly spliced protein, generated by a 2.2 kb doubly spliced hepatitis B virus RNA, is a pleiotropic activator protein mediating its effects via activator protein-1- and CCAAT/enhancer-binding protein-binding sites.

The 2.2 kb doubly spliced defective hepatitis B virus (HBV) genome is frequently detected in the serum of patients with chronic hepatitis B. However, the biological significance of this type of defective genome is not well understood. In this study, expression of the hepatitis B doubly spliced protein (HBDSP) was confirmed from the 2.2 kb doubly spliced defective HBV genome, which was isolated and transfected into Huh-7 hepatoma cells. To explore the potential pathogenicity of HBDSP, hepatocellular proteins interacting with HBDSP were screened by a yeast two-hybrid assay. Unexpectedly, HBDSP could transactivate the GAL4-responsive element, and deletion mapping revealed that the fragment located between residues Leu-48 and Gln-75 of HBDSP was crucial for transactivation activity. In Huh-7 hepatoma cells, HBDSP localized predominantly to the cytoplasm and showed transactivating effects on the cytomegalovirus immediate-early promoter, simian virus 40 enhancer/promoter and HBV regulatory elements including the S1 promoter, S2 promoter, Enhancer I and core upstream regulatory sequences. Further studies revealed that the transactivating activities were mediated by activator protein-1- and CCAAT/enhancer-binding protein-binding sites. These findings suggest that HBDSP is a pleiotropic activator protein that can potentially serve as an HBV virulence factor.