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.     

Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner.

The activities of the individual hepatitis B virus (HBV) promoters and the effects of the HBV enhancer on these promoters in several human cell types have been compared by measuring the activity and RNA levels of the linked reporter function chloramphenicol acetyltransferase. The relative promoter activities in the human HepG2 (liver), HeLa, and HS27 (fibroblast) cell lines are in the order precore greater than X greater than preS2 greater than preS1; thus, the promoters of the gene producing the largest quantity of viral proteins have relatively low activity. The juxtaposition of the HBV enhancer in either orientation increased the promoter activities only modestly (2- to 5-fold) in the nonliver cell lines, whereas it dramatically increased (20- to 100-fold) the promoter activities in the liver cell line. Thus, the HBV enhancer is especially active in liver cells. This may be one of the causes of hepatotrophicity of the virus.     

Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein

The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBV-infectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC50) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis Beta.     

Transcriptional activation of gammaherpesviral oncogene promoters by the hepatitis B viral X protein (HBx)

The latent membrane protein-1 (LMP1) of Epstein-Barr Virus (EBV), saimiri transformation protein (STP) of Herpesvirus saimiri (HVS), and K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) are potent gammaherpesvirus oncogenes. To study the possible effects of double viral infection, we investigated the effects of oncogenic early proteins of DNA viruses E1A and E1B (adenovirus-5), E6 and E7 (human papillomavirus-16), HBx (hepatitis B virus), Tag (SV40), and gammaherpesviral oncogene during co-infection in human B-lymphoma (Ramos) and human T-cell leukemia (Jurkat) cell lines. HBx transactivated the promoters of LMP1, STP, and K1 the most, by about six-, three-, and twofold, respectively. Analyses of site-directed mutation and the heterologous promoter system showed that HBx activated the promoter activity of these genes via the NF-kappaB site. These results suggest that HBV (HBx) infection of cells previously infected by gammaherpesviruses transactivates their oncogenes, resulting in possible virus-related disease pathogenesis.