An in vitro system for the production of hepatitis B virus (HBV) particles was established by the transient expression of transfected HBV DNA using a human hepatocellular carcinoma cell, HuH-7, as a recipient. The 3.6-and 2.2-kilobase transcripts observed were similar to those in virus-infected liver cells. Both transcripts revealed the microheterogeneity of their 5' ends. The formation of virusrelated particles subsequent to the RNA transcription was demonstrated. The core particles observed in the cytoplasm and the virus particles secreted in the culture medium contained the replicative intermediates of HBV DNA and banded at densities of 1.35-1.36 g/cm3 and 1.22-1.24 g/cm3, respectively. Furthermore, the in vitro mutagenesis of the template HBV DNA demonstrated that the P gene as well as the C gene products were essential for the production of HBV particles.Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis. Furthermore, HBV is known to be a major cause of human liver cancer. HBV DNA has the following unique structural characteristics (1) (2). However, the restricted host range of HBV and lack of a cell culture system for virus production have been major problems hindering from understanding of HBV replication and transcription at the molecular level.In this investigation, an in vitro system for the production of HBV particles was established by the transient expression of transfected HBV DNA, using a human hepatocellular carcinoma cell as a recipient. The RNA transcription of HBV DNA, similar to that in virus infection in vivo, was observed. Furthermore, mutagenesis of the template HBV DNA indicated virus production to be absolutely dependent on the template plasmid and the P gene as well as the C gene products to be essential for the synthesis of HBV DNA. MATERIALS AND METHODSCell Lines and DNAs. HuH-7 (3), huH2-2 (4), and HLEC-1 (5) were derived from human hepatocellular carcinomas. HuH-7 and HLEC-1 were negative for HBV infection and also for the integrated HBV DNA. HepG2 (6) was derived from hepatoblastoma and was negative for HBV integration.HuL-1 (7) was an established cell line derived from human fetal liver.Cloned HBV DNA (pHBV1-1) used for the construction of two different plasmids (Fig. 1) and DNA transfection was from the subtype adr virions, whose nucleotide sequence has been published (8). The first plasmid, pHBV-dimer, contains tandemly repeated sequences of 3.2-kb BamHI-digested whole genome at the BamHI site of pBR322. The second plasmid, pHBV-2, was derived from pHBVX-1, in which a 0.87-kb Stu IlBgl II fragment of HBV DNA was inserted into the BamHI site of pBR322 using a Bgl II linker DNA. The 3.2-kb BamHI fragment of the HBV genome was then inserted into the BamHI site of pHBVX-1 in the same orientation as that of the insert of pHBVX-1, resulting in the 4.1-kb insert of HBV DNA with 0.87-kb terminal repeats including the cohesive end region. Various mutant plasmids were prepared from pHBV-2 by the insertion of 8-base-pair (bp) Sac I linker DNA into the sites of ...

Toll-like receptors (TLRs

To examine the transforming potential of the × gene product of hepatitis B virus (HBV), the X‐gene‐containing region (referred to as the HBx region) was introduced into mouse NIH3T3 cells. Each transformed cell line expressed X‐coding mRNA at a different level. A positive correlation was found between the level of X‐coding mRNA and the saturation density of the cells. The HBx‐transformed cell lines exhibited × protein production and tumor formation in nude mice. The function of HBV in oncogenesis may involve the continuous expression of the X‐gene‐coded product in the HBV DNA‐integrated cells.

The integrated form of hepatitis B virus (HBV) in the human hepatoma cell line huH2-2 and its cellular counterpart sequence have been cloned and analyzed. Blot hybridization analysis and nucleotide sequencing indicated that a single copy of the 1895-base-pair (bp) subgenomic region of HBV DNA, spanning from the middle of pre-S to the end of gene X, was integrated and flanked by the 12-bp directly repeating cellular sequences. A comparison of the sequencing data with that of the cellular counterpart DNA indicated the absence of deletion and rearrangement in the cellular flanking DNA following integration of the 1895-bp HBV DNA, except for generation of the 12-bp direct repeat at the virus-cell junction. A possible model for the mechanism of HBV integration is proposed. Persistent hepatitis B virus (HBV) infection is etiologically related to primary hepatocellular carcinoma (1). In many cases of hepatocellular carcinoma, integration of HBV DNA has been demonstrated and some integrated forms of HBV DNA have been analyzed by molecular cloning (2-6). From analyses of their structural features, integrated viral sequences were found to be extensively rearranged (3, 4) as well as cellular flanking sequences in conjunction with that of the viral genome (6). The reason and mechanism of the extensive rearrangement of the integrated HBV genome and cellular flanking sequences are yet to be determined. However, these arrangements induced by integration of HBV DNA are suspected to give rise to some aberrant effects on the oncogene(s) and may play a part in causing liver tumors.In this investigation we analyzed thoroughly the integrated form of HBV DNA and its cellular counterpart in the human hepatoma cell line huH2-2. A single copy of the 1895-basepair (bp) subgenomic region was integrated into the chromosomal DNA. Interestingly, integration of HBV DNA was found to cause no extreme genetic change in the host chromosome(s), except that insertion of the viral genome generated a direct repeat at the virus-cell junction. One possible model for the mechanism of HBV integration is proposed and a correlation between HBV integration and liver carcinogenesis is discussed.MATERIALS AND METHODS DNA. The human hepatoma cell line huH2-2 used in this study was derived from the cell line huH2, previously established from the hepatocellular carcinoma of a 58-yearold patient in Japan (7). The serological markers were positive for the antibody toward hepatitis B core antigen (HBcAg) and negative for the hepatitis B surface antigen (HBsAg) and antibody toward HBsAg. Cultured cells were pelleted, followed by DNA extraction (8).Blot Hybridization. Blot hybridization was performed according to the method of Southern (9). The 32P-labeled hybridization probe was made by nick-translation (10).Cloning. DNA from huH2-2 cells was digested to completion with HindIII or BamHI, and the relevant fragments were partially purified by preparative agarose gel electrophoresis. As cloning vectors, Charon 28 for the BamHI fragments and Charon 21AM, a mod...

During pituitary development, the homeo domain protein GHF-1 is required for generation of somatotropes and lactotropes and for growth hormone (GH) and prolactin (PRL) gene expression. GHF-1 mRNA is detectable several days before the emergence of GH-or PRL-expressing cells, suggesting the existence of a somatotropic progenitor cell in which GHF-1 transcription is first activated. We have immortalized this cell type by using the GHF-1 regulatory region to target SV40 T-antigen (Tag) tumorigenesis in transgenic mice. The GHF-Tag transgene caused developmental entrapment of somatotropic progenitor cells that express GHF-I but not GH or PRL, resulting in dwarfism. Immortalized cell lines derived from a transgenic pituitary tumor maintain the characteristics of the somato/lactotropic progenitor in that they express GHF-1 mRNA and protein yet fail to activate GH or PRL transcription. Using these cells, we identified an enhancer that activates GHF-1 transcription at this early stage of development yet is inactive in cells representing later developmental stages of the somatotropic lineage or in other cell types. These experiments not only demonstrate the potential for immortalization of developmental progenitor cells using the regulatory regions from cell type-specific transcription factor genes but illustrate the power of such model systems in the study of developmental control.