Hepatitis B virus (HBV) possesses a 3.2-kb partially double-stranded DNA genome that is generated inside the nucleocapsid by the reverse transcription of the 3.5-kb pregenomic viral transcript. The initial steps in viral replication involve the recognition of an encapsidation signal termed epsilon (ε) at the 5'-end of the pregenomic RNA by the HBVpolymerase. The polymerase-bound pregenomic RNA is subsequently incorporated into an immature nucleocapsid particle and minus-strand HBVDNA synthesis is initiated utilizing the bulge region of ε as a template and a tyrosine residue in the amino-terminal region of the polymerase as a primer. Three nucleotides complementary to the 3'-end of the bulge region of ε are synthesized and subsequently translocated with the polymerase molecule to the acceptor site located in the DR1 sequence present at the 3'-end of the pregenomic RNA. Using mutagenesis analysis, a sequence element designated phi (ε) located upstream of the 3 ε DR1 sequence has been identified that is complementary to ε and is important for efficient viral replication. This element may bring the 3' DR1 sequence into proximity with the three nucleotide primer synthesized at the bulge of ε and facilitate primer translocation to the 3' DR1 acceptor sequence. Sequence elements with similar proximity to the 3' DR1 sequences and complementarity to ε are present in the woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV), suggesting the ε regulatory element may be phylogenetically conserved due to its functional importance in hepadnavirus minus-strand DNA synthesis.

The role and functional domain of hepatitis B virus (HBV) X protein (HBx) in regulating HBV transcription and replication were investigated with a transient transfection system in the human hepatoma cell line HepG2 using wild-type or HBx-minus HBV genome constructs and a series of deletion or mutation HBx expression plasmids. We show here that HBx has augmentation effects on HBV transcription and replication as a HBV mutant genome with defective X gene led to decreased levels of 3.5-kb HBV RNA and HBV replication intermediates and that these decreases can be restored by either transient ectopic expression of HBx or a stable HBx expression cell line. The C-terminal two-thirds (amino acids [aa] 51 to 154), which contain the transactivation domain, is required for this function of HBx; the N-terminal one-third (aa 1 to 50) is not required. Using the alanine scanning mutagenesis strategy, we demonstrated that the regions between aa 52 to 65 and 88 to 154 are important for the augmentation function of HBx in HBV replication. By the luciferase reporter gene analysis, we found that the transactivation and coactivation activities of HBx coincide well with its augmentation function in HBV transcription and replication. These results suggest that HBx has an important role in stimulating HBV transcription and replication and that the transcriptional transactivation function of HBx may be critical for its augmentation effect on HBV replication.

Hepatotropism is a prominent feature of hepatitis B virus (HBV) infection. Cell lines of nonhepatic origin do not independently support HBV replication. Here, we show that the nuclear hormone receptors, hepatocyte nuclear factor 4 and retinoid X receptor a plus peroxisome proliferator-activated receptor a, support HBV replication in nonhepatic cells by controlling pregenomic RNA synthesis, indicating these liver-enriched transcription factors control a unique molecular switch restricting viral tropism. In contrast, hepatocyte nuclear factor 3 antagonizes nuclear hormone receptormediated viral replication, demonstrating distinct regulatory roles for these liver-enriched transcription factors.

The nuclear hormone receptors hepatocyte nuclear factor 4 (HNF4) and the retinoid X a (RXRa) plus the peroxisome proliferator-activated receptor a (PPARa) heterodimer support hepatitis B virus (HBV) replication in nonhepatoma cells. Hepatocyte nuclear factor 3 (HNF3) inhibits nuclear hormone receptor-mediated viral replication. Inhibition of HBV replication by HNF3a is associated with the preferential reduction in the level of the pregenomic RNA compared with that of precore RNA. Hepatitis B e antigen (HBeAg), encoded by the precore RNA, mediates part of the inhibition of viral replication by HNF3a. The amino-terminal transcriptional activation domain of HNF3a is essential for the inhibition of HBV replication. The activation of transcription by HNF3 from HBV promoters downstream from the nucleocapsid promoter appears to contribute indirectly to the reduction in the steady-state level of 3.5-kb HBV RNA, possibly by interfering with the elongation rate of these transcripts. Therefore, transcriptional interference mediated by HNF3 may also regulate HBV RNA synthesis and viral replication.

Hepadnavirus replication occurs in hepatocytes in vivo and in hepatoma cell lines in cell culture. Hepatitis B virus (HBV) replication can occur in nonhepatoma cells when pregenomic RNA synthesis from viral DNA is activated by the expression of the nuclear hormone receptors hepatocyte nuclear factor 4 (HNF4) and the retinoid X receptor a (RXRa) plus peroxisome proliferator-activated receptor a (PPARa) heterodimer. Nuclear hormone receptor-dependent HBV replication is inhibited by hepatocyte nuclear factor 3 (HNF3). In contrast, HNF3 and HNF4 support duck hepatitis B virus (DHBV) replication in nonhepatoma cells, whereas the RXRa-PPARa heterodimer inhibits HNF4-dependent DHBV replication. HNF3 and HNF4 synergistically activate DHBV pregenomic RNA synthesis and viral replication. The conditions that support HBV or DHBV replication in nonhepatoma cells are not able to support woodchuck hepatitis virus replication. These observations indicate that avian and mammalian hepadnaviruses have distinct transcription factor requirements for viral replication.

A natural hepatitis B virus (HBV) variant associated with seroconversion from HBeAg to anti-HBe antibody contains two nucleotide substitutions (A1764T and G1766A) in the proximal nuclear hormone receptor binding site in the nucleocapsid promoter. These nucleotide substitutions prevent the binding of the retinoid X receptor a (RXRa)-peroxisome proliferator-activated receptor a (PPARa) heterodimer without greatly altering the efficiency of binding of hepatocyte nuclear factor 4 (HNF4) to this recognition sequence. In addition, these nucleotide substitutions create a new binding site for HNF1. Analysis of HBV transcription and replication in nonhepatoma cells indicates that RXRa-PPARa heterodimers support higher levels of pregenomic RNA transcription from the wild-type than from the variant nucleocapsid promoter, producing higher levels of wildtype than of variant replication intermediates. In contrast, HNF4 supports higher levels of pregenomic RNA transcription from the variant than from the wild-type nucleocapsid promoter, producing higher levels of variant than of wild-type replication intermediates. HNF1 can support variant virus replication at a low level but is unable to support replication of the wild-type HBV genome. These observations indicate that the replication of wild-type and variant viruses can be differentially regulated by the liver-specific transcription factors that bind to the proximal nuclear hormone receptor binding site of the nucleocapsid promoter. Differential regulation of viral replication may be important in the selection of specific viral variants as a result of an antiviral immune response.

An interferon-stimulated response element (ISRE)/interferon regulatory element (IRE) spanning nucleotide coordinates 1091-1100 is present in the enhancer 1/X gene promoter region of the hepatitis B virus (HBV) genome. In the context of a minimal promoter element, the enhancer 1/X gene promoter ISRE/IRE was shown to be a functional regulatory site capable of mediating interferon α-(IFNα) and interferonstimulated gene factor 3 (ISGF3)-specific transcriptional activation in transient transfection analysis. The enhancer 1/X gene promoter ISRE/IRE was also shown to mediate interferon regulatory factor (IRF) 1 and IRF7 activation of transcription from a minimal promoter construct. In contrast, IFNα and the IRFs had minimal effect on HBV transcription and replication in the context of the viral genome in cell culture.

A hepatitis B virus (HBV) transgenic mouse containing a naturally occurring mutation in the nucleocapsid promoter (A1764T plus G1766A) that inhibits the retinoid X receptor a (RXRa) plus peroxisome proliferator-activated receptor a (PPARa) heterodimer from binding to the proximal nuclear hormone receptor recognition sequence has been generated. Viral transcription and replication occur in the liver and kidney. The nucleocapsid promoter mutation does not prevent peroxisome proliferators from increasing viral transcription and replication in the liver of these variant HBV transgenic mice. This suggests that peroxisome proliferators may enhance viral transcription directly in a PPARa-dependent manner through the nuclear hormone receptor recognition site in the enhancer 1 region of the HBV genome. Hepatocyte nuclear factor 4 (HNF4) binding to the proximal nuclear hormone receptor recognition sequence in the nucleocapsid promoter appears to limit RNA synthesis from the precore transcription initiation site. Consequently, the variant HBV transgenic mice transcribe very little precore RNA and secrete extremely low levels of hepatitis B e antigen (HBeAg) compared with the wild-type HBV transgenic mice. This is consistent with the suggestion that viruses expressing HBeAg are preferentially eliminated in infected individuals when they seroconvert from HBeAg positive to anti-HBe antibody-positive status, leaving escape HBV variants that have reduced HBeAg expression.

AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably.

Transactivation of viral and host genes expression by hepatitis B virus X protein (HBx) is believed to be involved in hepatocarcinogenesis. The interaction of HBx with the tumor suppressor p53 and its inhibitory effect on p53 functions have been reported recently. However, the question of whether p53 is directly involved in EIBx transactivation has not yet been addressed. In this study, we delineated the interaction sites of HBx and p53 using far-Western blotting and glutothione S-transferase-resin pulldown assays. The results indicate that the HBx-binding sites are located within the oligomerizafion and specific DNA-binding domains of p53 and that the p53-binding site was confined to a small region in the HBx transactivatton domain. Mutual interference of the transactivations by HBx and p53 was detected by CAT assays in a transient transfection system. Strikingly, transactivation by HBx was observed in the p53-negative cells, Saos-2 and Hep3B, indicating that the transactivation and the p53-inhibiting functions of HBx are mutually interfering but distinct.