IDENTIFICATION OF FACTOR-BINDING SITES IN THE DUCK HEPATITIS-B VIRUS ENHANCER AND IN-VIVO EFFECTS OF ENHANCER MUTATIONS

Hepatitis B viruses (hepadnaviruses) can cause chronic, productive inf ections of hepatocytes. Analyses of the enhancers and promoters of the se viruses in cell lines have suggested a requirement of these element s for liver-enriched transcription factors. In this study, a minimum o f seven factor-binding sites on the duck hepatitis B virus enhancer we re detected by DNase I footprinting using duck liver nuclear extracts. Among the sites that were tentatively identified were one C/EBP-, one HNF1-, and two HNF3-binding sites. Mutations of the HNF1- and HNF3-li ke sites, which eliminated factor binding, as assessed by both DNase I footprinting and competitive gel shift assays, were evaluated for the ir effects on enhancer activity. Using a construct in which human grow th hormone was expressed from the viral enhancer and core gene promote r, we found that all of the mutations, either alone or in combination, reduced expression two- to fourfold in LMH chicken hepatoma cells. Th e mutations in the HNF1 site and one of the HNF3 sites, when inserted into the intact viral genome, also suppressed virus RNA synthesis in p rimary hepatocyte cultures. Virus carrying the latter HNF3 mutation wa s also examined for its ability to infect and replicate in ducks. No s ignificant inhibition of virus replication was observed in a short-ter m assay; however, virus with the HNF3 mutation was apparently unable t o grow in the pancreas, a second site of duck hepatitis B virus replic ation in the duck.