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Box alpha is an essential element of both the upstream regulatory sequence of the core promoter and the second enhancer, which positively regulate the transcription of human hepatitis B virus (HBV) genes. In this paper, we describe the cloning and characterization of a box alpha binding protein, E4BP4. E4BP4 is a bZIP type of transcription factor. Overexpression of E4BP4 represses the stimulating activity of box alpha in the upstream regulatory sequence of the core promoter and the second enhancer in differentiated human hepatoma cell lines. E4BP4 can also suppress the transcription of HBV genes and the production of HBV virions in a transient-transfection system that mimics the viral infection in vivo. Expression of an E4BP4 antisense transcript can, instead, elevate the transcription of the core promoter. A low abundance of E4BP4 protein and mRNA in differentiated human hepatoma cell lines is detected, and E4BP4 is not a major component of box alpha binding proteins in untransfected differentiated human hepatoma cell lines. C/EBPalpha and C/EBPbeta, in contrast, are major components of the box alpha binding activity present in nuclear extracts. E4BP4 has a stronger binding affinity towards box alpha than the endogenous box alpha binding activity present in nuclear extracts. Structure and function analysis of E4BP4 reveals that DNA binding activity is sufficient to confer the negative regulatory function of E4BP4. These results indicate that binding site occlusion is the mechanism whereby E4BP4 suppresses transcription in HBV.

Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.

The regulation of transcription of the hepatitis B virus core promoter is an important event in the viral life cycle. Two messages, precore and pregenomic RNAs, that are initiated 30 nucleotides apart are produced by the core promoter. Precore RNA encodes nucleocapsid protein and pregenomic RNA core and polymerase. The latter transcript also serves as a template for viral genome replication via reverse transcription. We have previously defined a basal core promoter, which contains four TA-rich sequences (TA1 through TA4) but no canonical TATA element, that can direct transcription of both messages. In this study, we demonstrated that a stretch of 15 nucleotides containing TA4 is sufficient to direct precise initiation of both precore and pregenomic transcripts. This sequence can function as both an initiator and a TATA element. Mutational analysis further revealed that sequences essential for either function are colocalized. The significance of this finding with respect to the basal transcription mechanism and regulation of viral gene expression is discussed.

Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation-dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined effects of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression.

Hepatitis B virus is a hepatotropic virus. Its replication and gene expression are mainly restricted to hepatocytes in the infective process. The viral gene expression thus provides a unique system with which to study the control of tissue-specific gene expression. We have previously reported the identification and characterization of the second enhancer (enhancer II) of hepatitis B virus. In this report, we further demonstrate that the minimal functional constituents of the second enhancer, box alpha and box beta, display liver cell and differentiation state specificity. Moreover, box alpha exhibits the same liver cell and differentiation state specificity when functioning as an upstream regulator for the basal core promoter. Gel shift experiments reveal a unique box alpha-binding protein, protein a, which is present only in differentiated liver cells, where enhancer II is functional. The converse is true for another box alpha-binding protein, protein f, which is present only in poorly differentiated liver cells and nonliver cells. The simplest hypothesis that explains these results is that protein a activates and/or protein f suppresses the enhancer and upstream regulator functions. Although C/EBP is a candidate for a transcription factor that interacts with box alpha or box beta, none of the binding factors identified in the gel shift assays, including protein a and protein f, is likely to be C/EBP because they differ from C/EBP in heat lability and sequence preference.

Hepatitis B virus (HBV) infection, either acute or chronic, has been one of the leading health problems in the world. To understand the HBV life cycle and disease process, we set out to study the regulation of viral gene expression. In this paper, we report the characterization of the HBV core promoter: two 3.5-kb transcripts, precore and pregenomic, are made from it. The latter is itself a template for viral genome replication and also encodes viral proteins essential for both viral replication and virion assembly. We identify a short sequence (from nucleotides [nt] 1744 to 1851, referred to as the basic core promoter [BCP]) that is sufficient to direct correct initiation of both precore and pregenomic messages. In addition, the two appear to be regulated in a coordinate manner. Sequences upstream of the BCP (from nt 1636 to 1744, referred to as the core upstream regulatory sequence [CURS]), have a strong stimulating effect on the BCP. Addition of the CURS to the BCP leads to a dramatic increase in both the transcription of two 3.5-kb messages and the production of 42-nm virions from transiently transfected hepatoma cells. The CURS stimulates the BCP in a position- and orientation-dependent manner. Therefore, it is unlikely that the effect is mediated through enhancer II, which has been localized to the same sequence. Deletion analysis of the CURS suggests that it contains multiple regulatory elements that control the BCP in an interactive manner. In accord with this hypothesis, the CURS is found to be bound with many distinct protein factors in footprinting experiments. Among these elements, box alpha (from nt 1646 to 1668) and box gamma delta (from nt 1671 to 1703) are two regulatory elements which individually stimulate promoter activity more than 100-fold.

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.

Previous studies have identified an enhancer (enhancer I) at nucleotides (nt) 1074 to 1234 in the genome of the human hepatitis B virus (HBV), which locates immediately upstream from the X gene. By analysis of the expression of the chloramphenicol acetyltransferase gene driven by a heterologous simian virus 40 early promoter, we describe the identification of a second enhancer (enhancer II) at nt 1636 to 1741, which locates downstream of enhancer I and immediately upstream of the core gene. With various deletions at the 5' end of enhancer II, a positive regulatory element was identified at nt 1636 to 1690 (the II-A element), with the 5' boundary between nt 1636 and 1671. The II-A element alone did not have an enhancer function, but the enhancer activity was achieved by the concomitant presence of the sequence from nt 1704 to 1741 (the II-B element). The II-B element alone did not have enhancer activity. These results indicate that cooperation between the II-A and II-B elements is required to exhibit the enhancer activity of enhancer II. We also show that enhancer II stimulates the transcriptional activity of both the SPI and SPII promoters of the surface gene. Therefore, the SPI promoter activity is regulated by the proximal HNF-1 binding element and the distal enhancers I and II. These results indicate that multiple regulatory elements scattered over the whole viral genome are involved in the regulation of expression of each individual HBV gene and that the same regulatory element controls the expression of different HBV genes. The relative positions of these regulatory elements in the HBV genome suggest that they may control the expression of HBV genes in a coordinate and cooperative manner.