Structure and expression of integrated hepatitis B virus genes in an HBs antigen producing human cell line (huGK-14)

A human continuous cell line (huGK-14) within a lineage of passaged cultures was investigated in the mode of integration and expression of hepatitis B virus (HBV) genes. HBV DNA was integrated in eight different sites of the cellular DNA, in each of which HBV genome was rearranged, fragmented, and/or partly deleted. Complete HBV genome that may lead to production of infectious virus particles was not detected in the cells nor in the culture medium. Clones of cDNA containing a complete coding frame for small HBs antigen protein (type adr) were obtained from mRNA of the cells. The cells were stable over the period of six months of cultivation and more than 60 population doublings in the mode of HBV integration and HBs mRNA expression. These results provide substantial evidence for the absence of an ability for the integrated DNA to create an infectious product in the cell; for the stable production of HBs mRNA from the cells, and suggest the usefulness of this cell line as a substrate for HBV vaccine production. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential cellular gene expression induced by hepatitis B and C viruses

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses.     

A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee.

The infectivity of hepatitis B virus (HBV) produced in vitro by HepG2 cells transfected with HBV DNA (HepG2T14) has been assayed in a chimpanzee. Following inoculation, the chimpanzee underwent a typical course of type B hepatitis infection, characterized by elevation of serum aminotransferases and by histological identification of hepatic damage. Hepatitis B surface antigen and core-related antigen appeared in the serum at weeks 5 and 7, respectively, after infection. HBV DNA was detected in serum samples, and replicative forms of the HBV genome were identified in liver biopsies. Subtype identification of hepatitis B surface antigen and restriction enzyme analysis of HBV DNA in both the inoculum and the serum of the infected chimpanzee confirmed that the hepatitis B infection observed in this animal was caused by viral particles produced by HepG2T14 cells. These findings indicate that, although HepG2 cells do not seem to be susceptible to infection by HBV in vitro, they can produce biologically active infectious virions after transfection with cloned HBV DNA.     

Hepatitis B virus X protein stimulates viral genome replication via a DDB1-dependent pathway distinct from that leading to cell death

The hepatitis B virus (HBV) X protein (HBx) is essential for virus infection and has been implicated in the development of liver cancer associated with chronic infection. HBx can interact with a number of cellular proteins, and in cell culture, it exhibits pleiotropic activities, among which is its ability to interfere with cell viability and stimulate HBV replication. Previous work has demonstrated that HBx affects cell viability by a mechanism that requires its binding to DDB1, a highly conserved protein implicated in DNA repair and cell cycle regulation. We now show that an interaction with DDB1 is also needed for HBx to stimulate HBV genome replication. Thus, HBx point mutants defective for DDB1 binding fail to complement the low level of replication of an HBx-deficient HBV genome when provided in trans, and one such mutant regains activity when directly fused to DDB1. Furthermore, DDB1 depletion by RNA interference specifically compromises replication of wild-type HBV, indicating that HBx produced from the viral genome also functions in a DDB1-dependent fashion. We also show that HBx in association with DDB1 acts in the nucleus and stimulates HBV replication mainly by enhancing viral mRNA levels, regardless of whether the protein is expressed from the HBV genome itself or supplied in trans. Interestingly, whereas HBx induces cell death in both HepG2 and Huh-7 hepatoma cell lines, it enhances HBV replication only in HepG2 cells, suggesting that the two activities involve distinct DDB1-dependent pathways.     

Interaction of aflatoxin and hepatitis B virus in the pathogenesis of hepatocellular carcinoma

Aflatoxin-B1 (AFB) and chronic hepatitis B virus (HBV) infection epidemiologically correlate with the geographic distribution of hepatocellular carcinoma (HCC). Integration of HBV DNA into the cellular genome of HCCs and the in vivo formation of adducts between AFB and nucleic acids lead us to suggest that hepatocytes with integrated HBV DNA preferentially accumulate AFB; the AFB-adducts formed may then initiate cell transformation by modifying the expression of critical host genes. The altered molecular biology of liver cells in HCC is evidenced by the fact that HBV does not replicate in HCC tissues or cell lines. The effect of AFB on the expression of cellular genes such as endogenous retrovirus(es) and possibly cellular oncogene(s) can be analyzed in HCC cell lines with and without integrated HBV DNA. In addition, human HCC tissues can be probed for HBV sequences and AFB-DNA adducts at the single-cell level. The presence of HBV and AFB can be correlated with the expression of putative transforming genes, providing a new insight into the interaction between liver cells, HBV and AFB in the pathogenesis of HCC.     

Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA.

DNA of hepatitis B virus is found to be integrated into the genome of infected human liver cells and may be related to the development of primary liver carcinoma. We have previously reported the cloning of cellular DNA with integrated HBV sequences from the PLC/PRF/5 cell line which derives from a human primary liver carcinoma. Two clones, designated as A-10.7 and A-10.5, and a third uncloned fragment are compared by restriction enzyme mapping, hybridization and nucleotide sequencing. The results indicate that amplification of integrated viral DNA and host flanking regions has occurred, followed by transposition and/or major deletions. The implications of these findings for the development of primary liver carcinoma are discussed.     

State of hepatitis B viral DNA in a human hepatoma cell line.

PLC/PRF/5, a tissue culture cell line isolated from a human hepatocellular carcinoma and producing hepatitis B surface antigen, was studied for the presence of hepatitis B virus (HBV)-specific DNA and RNA. PLC/PRF/5 cell DNA accelerated the rate of reassociation of HBV [32P]DNA, and quantitative experiments indicated that the cells contained approximately four copies of viral DNA per haploid, mammalian cell DNA equivalent. PLC/PRF/5 DNA accelerated the rate of reassociation of all individual restriction endonucleases HincII and HaeIII fragments of HBV [32P]DNA, indicating that DNA from all regions of the viral genome is present in the cells. This suggests that these cells contain at least most, and possibly all, of the viral genome. Digestion of PLC/PRF/5 cell DNA with restriction endonuclease HindIII (an enzyme found not to cleave the DNA of any HBV isolate so far examined) yielded only three fragments, all larger than virion DNA, which contained HBV DNA base sequences, suggesting that HBV DNA is integrated in high-molecular-weight DNA at three different sites in these cells and that there is no viral DNA in an episomal form. PLC/PRF/5 cell [32P]RNA was found to hybridize with all restriction fragments of HBV DNA adequately tested, indicating that at least most, and possibly all, of the viral DNA in these cells is transcribed.     

Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.

Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.-L. Chen, and G. Acs, Proc. Natl. Acad. Sci. USA 84:1005-1009, 1987) which elicit acute hepatitis in chimpanzees (G. Acs, M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper, Proc. Natl. Acad. Sci. USA 84:4641-4644, 1987). We report here the initial characterization of the viral nucleic acids produced in this culture system. Kinetic analyses of nuclear, cytoplasmic, and extracellular HBV DNAs were performed by Southern blotting with radiolabeled HBV strand-specific probes. The results from these analyses indicate that at the stationary cellular growth phase, there is a dramatic increase in the rate at which HBV DNA accumulates. Incomplete double- and single-stranded forms of the HBV genome were detected in the nuclear and cytoplasmic fractions as well as in the extracellular medium. In addition, the nuclear DNA apparently includes multiple complete copies of the HBV genome chromosomally integrated and full-length covalently closed circular HBV DNA. Multiple HBV-specific polyadenylated RNAs with lengths of 3.5, 2.5, and 2.1 kilobases were identified by Northern (RNA) blot analysis. S1 nuclease mapping and primer extension identified a single 3' end and multiple unique initiation sites corresponding to nucleotides just 5' to the pre-S1 region, as well as upstream and within the pre-S2 and precore regions. The nucleic acid profile obtained from these analyses is essentially a facsimile of that obtained by studying liver tissue from HBV-infected individuals.     

利用基因捕获技术建立稳定表达HBV的细胞模型

pGEM-HBV1.3质粒经HindIII限制性内切酶消化,将HBV1.3全长DNA切下,与同样经HindIII限制性内切酶降解过的PU21连接,得到PU21-HBV重组质粒。将该重组质粒采用电击转染方法导入HepG2细胞中,G418筛选阳性克隆并以X-gal染色,RT-PCR、Southern blot等方法验证HBV DNA的插入和表达。 PU21-HBV重组质粒经测序证明HBV1.3全长DNA正确与PU21载体连接,该重组质粒转染HepG2细胞后经G418筛选,得到一系列阳性克隆, Southern blot证实HepG2细胞基因组中含HBV DNA,RT-PCR结果表明HBV DNA在HepG2细胞中有功能基因的转录。HBV1.3已被整合在HepG2细胞染色体中并能稳定表达其RNA。稳定的HBV表达细胞模型构建成功。HBV表达细胞模型的建立,为进一步研究相关基因对HBV的转录、复制、转录后调节以及HBV各种蛋白的表达机理研究提供实验材料。     

Replication of hepatitis B virus which carries foreign DNA in vitro.

Targeting a specific DNA sequence to the desired tissues is an important step in gene therapy. The hepatitis B virus (HBV) is the only DNA virus that has hepatocyte specificity. We attempted to construct an HBV-based vector for targeting the liver. We observed the replication and secretion of virus particles in an HBV construct that lacks X gene and carries an extra 63 bp DNA fragment in vitro. Replication was observed in the cell line HuH-7 but not HepG2. From this construct, we designed an HBV-based vector that could carry foreign DNA. HBV based vectors provide for the possibilities of generating therapeutic agents for individual patients. Our host vector system may be used to clear out the HBV from the HBV carrier or chronic hepatitis B patients by introducing a genetically engineered HBV into these patients.     

Integration pattern of hepatitis B virus DNA sequences in human hepatoma cell lines.

Four human hepatoma cell lines established from primary hepatocellular carcinomas were examined for the presence of hepatitis B virus DNA sequences. Reassociation kinetic analysis indicated that the cell lines HEp-3B 217, HEp-3B 14, HEp-3B F1, and PLC/PRF/5 contained two, one, one, and four genome equivalents per cell, respectively. Southern blot hybridization analysis demonstrated that hepatitis B virus DNA was integrated into the cellular DNAs of these cell lines. Further liquid hybridization studies with 32P-labeled HincII restriction fragments of hepatitis B virus DNA established that DNA sequences from all regions of the HBV genome were represented in the integrated viral sequences. Although the three HEp-3B cell lines were derived from the same tumor, they differed significantly in their patterns of integration of hepatitis B virus DNA, the number of copies of viral DNA per cell, and their ability to produce the virus-coded surface antigen.     

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.     

iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

乙肝病毒基因组克隆及其线性分子侵染力研究

乙肝病毒基因组为环状双链DNA,常态环状分子侵染能力很强。为了探讨乙肝病毒基因组线性分子对人肝的侵染和复制能力,克隆了德国病人Brandt携带的乙肝病毒全长基因组,定名为HBV-Brandt。Multiplex PCR基因型鉴定表明HBV-Brandt属于乙型肝炎病毒A型。细胞转染和荧光定量PCR分析证明乙肝病毒HBV-Brandt的基因组线性DNA分子可以侵染HepG2细胞,并进行大量复制。     

A hepatocellular carcinoma cell line producing mature hepatitis B viral particles

Current in vitro models for hepatitis B virus (HBV) are based on human hepatoblastoma cell lines transfected with HBV genome. The objective of this work was to develop an in vitro, hepatocellular carcinoma (HCC)-based system supporting HBV full replication and producing mature viral particles. The FLC4 human HCC cell line was stably transfected with a plasmid carrying a head-to-tail dimer of the adwHBV genome. One of the clones, FLC4A10II, exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. Furthermore, the cells produced HBV particles that were detected by a cesium chloride density gradient performed on the culture medium. Analysis by Southern blot revealed that HBV DNA has integrated into the FLC4A10II cell genome. The presence of HBV in the FLC4A10II cells did not cause alterations in cell morphology and the cells continued to resemble mature hepatocytes. They do exhibit a high mitotic activity. The new HBV stably transfected cell line, FLC4A10II, can serve as an important tool for further exploration of HBV host-pathogen interaction, viral life cycle, and for assessing new antiviral agents.