Abstract
We have used a novel approach called expression selection to precisely define the hepatitis B virus (HBV) enhancer. Expression selection is based on a shuttle vector containing an enhancerless SV40 T antigen gene, the SV40 origin of replication and a plasmid replicon. This vector is linearized, ligated with the sonicated DNA to be analyzed and transfected into eukaryotic cells, where only plasmids which have incorporated an enhancer can express T antigen and therefore replicate. Vectors amplified by replication are selectively rescued in E. coli and their inserts analyzed. When we performed this protocol with HBV DNA we rescued two overlapping fragments of 166 and 214 bp which in HBV DNA map about 500 bp upstream of the core antigen mRNA initiation site and 1150 bp downstream of the surface antigen mRNA initiation site. These results were confirmed by conventional deletion mapping. When compared to the SV40 enhancer in nonhepatic cell lines, the HBV enhancer is only 5 to 10% as active; nevertheless, it also acts in an orientation-independent manner and in a position downstream of a gene. The HBV enhancer is situated in the coding region of the potential reverse transcriptase, and thus is the first enhancer identified to map in a protein-coding region.