A functional hepatocyte nuclear factor 3 binding site is a critical component of the duck hepatitis B virus major...

The gene coding for the S protein, the smaller of the two envelope antigens of the duck hepatitis B virus (DHBV), is transcribed from a TATA-less promoter. In this study, we localized the promoter to a 245-bp segment of the genome that was capable of efficiently driving expression of a linked reporter gene upon transient transfection into the differentiated hepatoma cell lines LMH and HepG2. However, no measurable activity from this construct could be detected in similar assays with the dedifferentiated cell line HepG2.1 or the nonhepatic cell line HeLa. Located at position -25 relative to the transcriptional start site was a sequence conforming to the consensus binding site for hepatocyte nuclear factor 3 (HNF3). Deletion of this region reduced activity of the reporter gene to barely detectable levels in LMH cells. The results of electrophoretic mobility shift analysis (EMSA) demonstrated that a double-stranded oligonucleotide containing this sequence formed a specific complex with DNA-binding proteins from LMH and HepG2 cells but not with nuclear extracts obtained from HepG2.1 or HeLa cells. Cotransfection of HepG2.1 cells with DHBV S promoter constructs and a rat HNF3beta expression plasmid resulted in transactivation of only those constructs in which the candidate HNF3 site was present. Furthermore, EMSA using HepG2.1 nuclear extracts containing exogenously expressed HNF3 formed complexes with the same migration and competition properties as those in which the proteins were derived from the differentiated hepatoma cells. Thus, several lines of evidence suggest a critical role for HNF3 in activity from the DHBV S promoter.