A negative regulatory element and its binding protein in the upstream of enhancer II of hepatitis B virus

The hepatitis B virus (HBV) core/pregenomic promoter is regulated by enhancer I (ENI) and enhancer II (ENII) which are located upstream of the initiation sites of core/pregenomic transcripts. In this study, we identified a negative regulatory element (NRE) (nt 1576 to 1639) upstream of ENII by serial deletion analysis; a 33 kDa cellular protein in HepG2 cells binds to this element. The NRE has a significant activity if it is located upstream of ENII in HepG2 cells. Mutational analysis showed that the sequence (5'-CCAC-3') from nt 1612 to 1615 is responsible for the repression activity of NRE. Southwestern blotting and UV-crosslinking assays with HepG2 nuclear extracts also demonstrated that the 33 kDa protein in HepG2 cells binds to the sequence. It, thus, appears that the 33 kDa protein is responsible for the repression activity of NRE.