Functional organization of the hepatitis B virus enhancer.

We have studied the functional constituents of the hepatitis B virus enhancer in a number of cell lines. The sequence of this enhancer, being embedded within an open reading frame of the virus, is in part evolutionarily frozen and therefore serves as a good model to investigate the fundamental enhancer elements. The hepatitis B virus enhancer contains three functionally important DNA sequence elements, EP, E, and NF-1a, each of which is bound by a distinct protein(s). The synergistic action of these elements accounts for all of the enhancer activity in a nonliver cell line and for most, but not all, of the activity in liver-derived cell lines. Multimers of the E but not of the EP element act as an autonomous enhancer. Conversely, a single element of either the E or the NF-1a element can act only when linked to the EP element. These results suggest that EP is a crucial enhancer element that acts only in interaction with a second enhancer element with intrinsic enhancer activity. Interestingly, a highly similar enhancer structure is found in a number of distinct viruses.     

Characterization of the hepatitis B virus EnhI enhancer and X promoter complex.

The hepatitis B virus EnhI enhancer element overlaps the promoter of the X gene. By performing methylation interference experiments, four protein factor binding sites clustered in a 120-bp region were found to control the EnhI enhancer and X promoter activities. Deletion mapping experiments indicated that the two upstream protein factor binding sites constituted a basal enhancer module. This module, likely bound by a liver-specific factor and a ubiquitous factor, could activate the herpes simplex virus thymidine kinase gene promoter by 5- or 10-fold, depending on the orientation, in Huh7 cells, a liver-derived cell line, but not in other cell types tested. The two downstream protein factor binding sites interact with the upstream basal enhancer module in an orientation- and distance-dependent manner to increase the enhancer activity by another 10-fold. In addition, at least one of the two downstream protein factor binding sites is also essential for the X promoter activity.     

The human hepatitis B virus enhancer requires trans-acting cellular factor(s) for activity.

The activity of the hepatitis B viral enhancer element was studied in various cell lines. This enhancer shows strict host and tissue specificity in that it is functional only in liver cells of human origin. Further, it requires trans-acting factor(s) present in liver cells for activity, and this activity is independent of hepatitis B virus gene products in the cell lines tested.     

Serological relationship of woodchuck hepatitis virus to human hepatitis B virus.

Two antigenic systems of the woodchuck hepatitis virus have been identified. The relationship between viral antigens of the woodchuck hepatitis virus and the human hepatitis B virus was determined by using immunoprecipitation, hemagglutination, and immune electron microscopy techniques. Antigens found on the cores of the two viruses were cross-reactive. Lack of cross-reactivity between the surface antigens of the two viruses in immunodiffusion experiments suggested that the major antigenic determinants of the viral surfaces are different; however, results of passive hemagglutination tests indicated that there are common minor determinants. Nucleic acid homology, as measured by liquid hybridization, was found to be 3 to 5% of the viral genomes. The results of this study provide further evidence that woodchuck hepatitis virus is the second member of a new class of viruses represented by human hepatitis B virus. Since virus-infected woodchucks may acquire chronic hepatitis and hepatocellular carcinoma, these antigens and their respective antibodies will be useful markers for following the course of virus infection in investigations of the oncogenic potential of this class of viruses. The nucleocapsid antigen described may be a class-specific antigen of these viruses and, thus, may be useful in discovering new members of the group.     

Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains.

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.     

Bioluminescence imaging of hepatitis B virus enhancer and promoter activities in mice

By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1-6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.     

Structural relationships between the surface antigens of ground squirrel hepatitis virus and human hepatitis B virus.

Several physical, chemical, and serological properties of surface antigen particles from ground squirrel hepatitis virus (GSHsAg) and human hepatitis B virus (HBsAg) were compared. GSHsAg and HBsAg particles were purified from positive sera by gel chromatography and isopycnic centrifugation. Both antigens consisted mainly of spherical particles with an average diameter of approximately 20 nm and a buoyant density in CsCl of approximately 1.19 g/ml. Their UV absorption spectra indicated the presence of more tryptophane than tyrosine and the absence of detectable nucleic acid. GSHsAg was found to contain two major polypeptides of approximately 23,000 and 27,000 daltons, with electrophoretic migration rates distinctly faster than those of the two major polypeptides of HBsAg particles. After radiolabeling of purified antigen preparations with Bolton-Hunter reagent, the two major polypeptides of GSHsAg showed almost identical tryptic peptide maps. The tryptic peptide map of the major polypeptide from GSHsAg contained 13 of 37 spots also present in the map of the major HBsAg polypeptide, and 13 of 27 spots in the map of the major HBsAg polypeptide were also present in the map of the major GSHsAg polypeptide. This suggests considerable sequence homology between the major surface antigen polypeptides of the two viruses. However, there was only a weak serological cross-reactivity between antigens of the two viruses. Using an anti-HBs-containing serum with a relatively strong cross-reactivity, GSHsAg was found to consist of at least two antigenically different subspecies. The more strongly cross-reacting from had a slightly higher buoyant density than the other antigenic form.