The S promoter of hepatitis B virus is regulated by positive and negative elements.

The S promoter, one of the major hepatitis B virus (HBV) promoters, directs the synthesis of mRNA for surface antigen. Transient expression studies revealed that this promoter is highly active in the Alexander hepatoma cell line but not in SK-Hep1 and HeLa cells. We found that a distal element of the promoter (-103 to -48) confers this cell-type-specific behavior through a mechanism in which the promoter activity is repressed in HeLa and SK-Hep1 cells but increased in Alexander cells. By using an inhibitor of protein synthesis, we obtained evidence that a labile repressor(s) confers the negative effect in SK-Hep1 cells. We also found an enhancerlike activity associated with a small DNA segment of the S promoter (-27 to + 30). This proximal element was active in HeLa and SK-Hep1 cells only in the absence of the distal negative element. Finally, analysis of S promoter deletion mutants demonstrated that the -27 to -17 region of the S promoter is crucial for its activity.     

High affinity binding site for nuclear factor I next to the hepatitis B virus S gene promoter.

The hepatitis B virus (HBV) surface antigen (HBsAG) is encoded by the S gene under the regulation of a promoter in the pre-S1 region. The S gene promoter does not contain a 'TATA' box-like sequence, but there is a sequence resembling, in part, the late promoter of Simian virus 40 (SV40). In an attempt to study the regulation of the S gene promoter we looked for cellular proteins which bind to this region. We report here that a nuclear protein is tightly bound to the HBV genome at a position approximately 190 bases upstream from the S gene promoter. Evidence is provided to show that (a) this nuclear protein is the nuclear factor I (NF-I) that was previously found to be bound to the inverted terminal repeat of the adenovirus (Ad) DNA and to enhance Ad DNA replication in vitro and (b) this NF-I binding site is required for optimal activity of the S gene promoter.     

Intracellular inhibition of hepatitis B virus S gene expression by chimeric DNA-RNA phosphorothioate minimized ribozyme

Chronic hepatitis B virus (HBV) infection is a major problem in Asia. Current therapies for chronic hepatitis B have limited efficacy. The successful use of ribozymes for intracellular inhibition of HBV gene expression was recently reported. As an alternative to ribozymes, the use of DNA-containing, phosphorothioate-modified, minimized hammerhead ribozymes (minizymes) to inhibit hepatitis B surface antigen (HBsAg) expression and viral replication was investigated. Such molecules can be synthesized and supplied exogenously. Two conserved sites within the HBsAg open reading frame (ORF) were targeted. PLC/PRF5 cells or 2.2.15 cells were treated with minizymes or antisense oligomers to assess the effects on cell viability, HBsAg expression, and viral DNA production. Treatment with the minizyme, MZPS1, resulted in >80% inhibition of HBsAg expression in PLC/PRF5 cells. MZPS1 had more inhibitory effect than the antisense oligonucletoide target at the same region, whereas the control minizyme had little effect. Another gene-specific minizyme, MZPS2, did not show any effect. Treated cells remained fully viable. Treatment of 2.2.15 cells with MZPS1 also led to decreased HBsAg expression. In addition, a 2.3-fold decrease in viral production was observed. Our data showed that minizymes can inhibit HBV gene expression and may potentially be useful for clinical therapy against chronic HBV infection.     

Identification of a promoter element located upstream from the hepatitis B virus X gene.

We have analyzed a series of plasmids in which the sequences located upstream from the hepatitis B virus (HBV) X gene were linked to the chloramphenicol acetyl transferase (CAT) gene. Expression of the marker CAT gene in transfected cells clearly demonstrated that sequences preceding the X gene contain an active promoter. RNA mapping by primer extension indicated that the RNA encoded by the X gene promoter initiates at multiple sites spanning nucleotides 1250 to 1350 on the HBV genome. Deletion within the adjacent HBV enhancer element region significantly reduced the activity of the X gene promoter, suggesting that the X gene promoter requires the enhancer element for maximal activity.     

Induction of hepatitis B virus gene expression at low temperature

There is a limited understanding of the cellular regulation of HBV gene expression in differentiated hepatocytes. We previously demonstrated that HBV replication inversely correlates with cell proliferation and DNA synthesis. In this report, temperature-induced modulation of cell growth was used as a novel approach to study HBV gene expression in the absence of indirect effects from drugs or serum deprivation. We observed markedly elevated levels of hepatic HBV mRNA expression from integrated and episomal HBV DNA at 32 degrees C. Additionally, hepatoblastoma cells cultured at 32 degrees C expressed increased levels of albumin mRNA and decreased levels of c-myc mRNA, which demonstrates that liver-derived cells cultured at low temperature exhibit characteristics of functional and differentiated hepatocytes. In transiently transfected HepG2 cells cultured at 32 degrees C, the HBV enhancer 1 activated the X promoter and core/pregenomic promoter by 7.3- and 28-fold, respectively. In the absence of enhancer 1, core/pregenomic promoter activity was 2.4-fold higher than the X promoter in HepG2 cells at 32 degrees C. In contrast, enhancer 1 exclusively activated the X promoter in transfected non-liver cells at 32 degrees C. Therefore, the core/pregenomic promoter exhibits strict liver-specificity at low temperature. This work supports the hypothesis that HBV replication and gene expression are optimal in non-activated hepatocytes, and provides a novel system for delineating molecular aspects of the HBV replication process.     

The xylA promoter of Bacillus megaterium mediates constitutive gene expression in Escherichia coli

In the present study, we demonstrate that the Escherichia coli–Bacillus megaterium shuttle vector pHIS1522 can be used as a versatile expression vector. Recombinant genes under the control of the xylA promoter are constitutively expressed at a high level in E. coli strains, whereas their expression is strongly induced by the addition of xylose in B. megaterium. The utilization of D ‐xylose is known to be dependent on the xylAB genes in a number of bacteria. For B. megaterium a XylA‐based expression system was established that allows tightly regulated and highly efficient heterologous gene expression. The open reading frame (ORF) of the fluorescent protein turboRFP was cloned under the control of the xylA promoter of B. megaterium in the shuttle vector pHIS1522. Unexpectedly, tRFP expression was not only observed in B. megaterium, but also in E. coli. Based on fluorescence measurements and Western blot analysis, expression was comparable or slightly higher compared with the commonly used pET vectors. Therefore, pHIS1522 can be used as a versatile expression vector in both, B. megaterium and E. coli.