The PreS2 activators of the hepatitis B virus: activators of tumour promoter pathways.

In addition to causing acute and chronic hepatitis, hepatitis B virus (HBV) is considered to be a major cliological factor in the development of human hepatocellular carcinoma (HCC). Epidemiological studies have demonstrated an approximately 10-fold increase in the relative risk of HCC among HBV carried compared to noncarriers. Almost all HBV-associated HCCs studied so far harbor chromosomally integrated HBV DNA. Integrated viral DNA can encode two types of transcriptional activators, the HBx protein and the PreS2 activators [the large surface proteins (LHBs) and truncated middle surface proteins (MHBs)]. The activator function of the PreS2 activators is based on the cytoplasmic orientation of the PreS2 domain. The PreS2 domain is PKC-dependent phosphorylated. Moreover, the PreS2 domain binds of PKC alpha/beta and triggers a PKC-dependent activation of the c-Raf-1/MAP2-kinase signal transduction cascade, resulting in an activation of transcription factors such as AP-1 and NF-kB. Furthermore, by activation of this signaling cascade, the PreS2 activators cause an increased proliferation rate of hepatocytes. According to the two-step model of carcinogenesis (initiation/promotion), the PreS2 activators could exert a tumour-promoter-like function by activation of the PKC/c-Raf-1/MAP2-kinase signaling cascade: cells harboring critical mutations (initiation) may be positively selected (promotion). Such a multistep process may account for the long latency period in HCC development, but it also leads to the hypothesis that each tumor reflects an individual case.

Isolation and molecular characterization of hepatitis B virus X-protein from a baculovirus expression system.

The X protein (HBx) of the human Hepatitis B Virus (HBV) is a regulatory protein that exercises a transcriptional activator function on a variety of regulatory elements and is therefore considered to be involved in the development of human hepatocellular carcinoma (HCC). So far, most attempts at elucidating HBx function have been undertaken at the genetic level, reflecting the difficulties in detecting the very low amounts of the protein in infected livers. Consequently, the questions of intracellular localization and posttranslational modification have not yet been completely answered. We therefore constructed recombinant baculoviruses that allowed expression of HBx and the hexa histidine HBx fusion protein HBxHis in insect cells. Cell fractionation experiments revealed that only a minor part of HBx is detectable in a soluble form in the cytosolic fraction, whereas most of the protein forms intracellular aggregates. These results could be confirmed by confocal laser immunofluorescence. The fusion of a hexa-histidine tag to the amino terminus of HBx allowed a rapid one-step purification by metal chelate affinity chromatography. The detailed analysis of purified HBxHis using electrospray ionization mass spectrometry uncovered two major components: the unmodified, monomeric, fully oxidized form with five intramolecular disulfide bridges, and its N-acetylated modification. Additionally, two minor peaks with mass differences of delta m = +80 da suggested that a small fraction of HBx becomes posttranslationally phosphorylated in insect cells. No further modifications could be observed, indicating that only phosphorylation might play a role in a possible posttranslational regulation of this viral activator.

Shock wave permeabilization as a new gene transfer method.

Uptake of naked functional DNA into mammalian cells can be achieved by a number of physical methods. However, for most of these techniques possibilities for therapeutic in vivo applications--especially to solid organs--are often limited. In this report, we describe shock wave permeabilization as a new physical gene transfer method, which can be easily applied, provides great flexibility in the size and sequence of the DNA molecules to be delivered, and which should exhibit an advantageous security profile in vivo. Upon exposure to lithotripter-generated shock waves eukaryotic cells display a temporary increase in membrane permeability. This effect was shown to be caused by cavitation resulting in the transient generation of cell pores which allows the direct transfer of naked plasmid DNA. Shockwave transfection of a variety of cell lines was demonstrated. Since shock waves can be well focused within particular body regions, future applications of extracorporally generated shock waves to tissues simultaneously perfused with DNA solutions might open up the possibility of achieving a regionally enhanced in vivo gene transfer.

The hepatitis B virus MHBst167 protein is a pleiotropic transactivator mediating its effect via ubiquitous cellular transcription factors.

C-terminally truncated surface proteins of hepatitis B virus (HBV) are frequently translated from genomically integrated viral sequences. They may be relevant for hepatocarcinogenesis by stimulating gene expression. First, we examined the transactivating potential of middle hepatitis B surface protein truncated at amino acid (aa) position 167 (MHBst167) on the HBV regulatory element. In transient cotransfection assays using Chang liver or HepG2 cell lines and chloramphenicol acetyltransferase (CAT) reporter constructs only the HBV enhancer I, but no other HBV regulatory elements like the X promoter, the S1 or S2 promoter or the enhancer II/core promoter could be stimulated by MHBst167. Since there is no evidence for a direct interaction of MHBst167 with DNA, we subsequently analysed whether cellular transcription factors were involved in mediating transactivation. This was tested both with isolated transcription-factor-binding sites and in the natural context of viral and cellular promoter elements. Deletion analysis and electrophoretic mobility shift assays revealed that Sp1, AP1 and NF-kappa B can mediate transactivation by MHBst167. No involvement of CREB, NF1 or the liver-specific factor C/EBP was found. These data indicate that MHBst167 is a pleiotropic, non-liver-specific transactivator which exerts its effect via ubiquitous cellular transcription factors that are also involved in the regulation of expression of cellular genes relevant for proliferation and inflammation.

Core specific antisense phosphorothioate oligodeoxynucleotides as potent and specific inhibitors of hepatitis C viral translation.

Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementary to sequence stretches in the 5' non coding region (NCR) of the hepatitis C virus (HCV) have recently been shown to effectively inhibit viral gene expression. In order to further delineate the optimum target region in the highly conserved 5' end of the viral RNA, S-ODN complementary to HCV core coding sequences were analysed in the present study. In a rabbit reticulocyte lysate (RRL) in vitro translation assay S-ODN 5, complementary to the HCV-RNA nucleotides 340-353, and S-ODN-6, complementary to nucleotides 348-365, resulted in an inhibition of viral translation of 90.4 +/- 1.3% and 93.7 +/- 5.1%, respectively at a concentration of 4.14 microM. S-ODN 7, complementary to nucleotides 371-388, was relatively inefficient and showed a maximal inhibition of 42.4 +/- 12.2%. It has been suggested that in living cells an inhibition by S-ODN is mainly mediated by the action of RNAse H. In RRL the RNAseH content is very low; therefore, to simulate the situation in living cells inhibition experiments in RRL enriched with RNAse H were performed. Under these conditions S-ODN 5, 6 and 7 inhibited viral translation by 45.6 +/- 6.3%, 80.3 +/- 2.8% and 70.9 +/- 5.7% at concentrations as low as 0.2 microM. At this concentration no inhibition was observed in the standard RRL assay. In cell culture S-ODN 7 was by far the most efficient inhibitor of viral translation, resulting in a specific inhibition of 89.4 +/- 3.6% at a concentration of 0.3 microM. Taken together with the results of our previous study, nucleotides 326-348 comprising the 3' end of the NCR and nucleotides 371-388, located entirely in the core coding region of the HCV RNA, are effective targets for S-ODN mediated inhibition of viral translation.

The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

Isolation of highly purified, functional carboxy-terminally truncated hepatitis B virus middle surface protein activators from eucaryotic expression systems.

Carboxy-terminally truncated hepatitis B virus (HBV) middle surface proteins (MHBst) show a transcriptional activator function. Two different subtypes of MHBst activators can be distinguished: an ER-localized type, represented here by MHBst76 (truncated at amino acid 76), and a cytosol-localized type, represented here by MHBst63. To characterize the MHBst activator on the protein level and to analyze posttranslational modifications, we established recombinant baculoviruses encoding for fusion proteins of MHBst76 or MHBst63 and of an amino terminal hexa-his tag. Both proteins could be obtained in high purity by affinity chromatography using Ni-nitrilo-tri-acetate agarose. In addition, 6H-MHBst76 was also isolated from transiently transfected HepG2 cells. Both the Spodoptera frugiperda (Sf9) cell-derived and the HepG2 cell-derived MHBst proteins were found to be unglycosylated. A detailed analysis of Sf9 cell-derived 6H-MHFBst76 by electrospray-ionization mass spectrometry showed that a fraction of this protein is N-terminally acetylated and phosphorylated or sulfated. Electric-field-mediated transfer of the highly purified proteins into reporter cells demonstrated that the isolated proteins are functional transcriptional activators. These experiments further showed that Sf9 cell-derived and HepG2 cell-derived 6H-MHBst do not differ in their functionality. This system allowed production and purification of functional 6H-MHBst in amounts sufficient enough to allow a further detailed analysis of MHBst activators on the protein level.

Anti-hepatitis B virus activity of N-acetyl-L-cysteine (NAC): new aspects of a well-established drug.

N-acetyl-L-cysteine (NAC) is commonly administered as an antidote against acetaminophen intoxication and is the preferred agent in the treatment of pulmonary diseases. It is furthermore commonly considered that it restrains human immunodeficiency virus (HIV) replication by scavenging reactive oxygen intermediates (ROI) and thus suppressing activation of nuclear factor kappa B (NF kappa B). We show here that NAC is in addition able to inhibit hepatitis B virus (HBV) replication, but by a mechanism independent of the intracellular level of reactive oxygen intermediates. Treatment of HBV-producing cell lines with NAC resulted in an at least 50-fold reduction of viral DNA in the tissue culture supernatant within 48 h. This decrease of viral DNA and thus of virions in the tissue culture supernatant is caused by a disturbance of the virus assembly, rather than by a reduction of viral transcripts. Our data strongly suggest a potential use of this well-established, non-toxic drug for the treatment of HBV infection. Since NAC, in contrast to interferon, exerts its anti-HBV activity at a posttranscriptional level, a combination of NAC with the established interferon therapy could also be considered.

Keratinocyte growth factor is highly overexpressed in inflammatory bowel disease.

Recently we demonstrated an important function of keratinocyte growth factor (KGF) in wound re-epithelialization. As KGF is mitogenic for various epithelial cells, we speculated about a role of KGF in epithelial repair processes of other organs as seen in a variety of inflammatory diseases. Here we demonstrate a strikingly increased expression of KGF in surgical specimens from patients suffering from Crohn's disease and ulcerative colitis. The levels of KGF expression strongly correlated with the degree of inflammation as assessed by histological analysis of adjacent tissue and expression analysis of the pro-inflammatory cytokine interleukin-1 beta. The highest levels of KGF mRNA and protein were found in mesenchymal cells of the lamina propria, particularly in highly inflamed areas. As the KGF receptor is expressed in intestinal epithelial cells, KGF seems to act in a paracrine manner to stimulate proliferation of these cells. These data suggest a crucial role of KGF in epithelial repair after injury caused by inflammatory processes.

Hepatoma-derived integrated HBV DNA causes multi-stage transformation in vitro.

The hepatoma-derived hepatitis B virus (HBV) DNA insert HU-a has recently been shown to contain two viral transactivator genes, X and preS2 /S. We report here that HU-a induces malignant transformation after stable transfection of the fetal mouse hepatocyte line FMH202, as indicated by soft agar growth and nude mouse tumorigenicity. Transfections with HU-a subclones, containing the X gene of the preS2 /S gene alone or sequences without transactivator gene, respectively, suggested that the X gene is essential for transformation. Sequential stages of transformation and tumor progression were analysed by injection of the stably transfected FMH202 lines into nude mice, explanation of the resulting tumors and re-establishment of cell lines from the tumors. Comparison of two HU-a-transformed cell lines by HBV mRNA hybridization, Southern analysis and chromosomal in situ hybridization revealed that integrated HBV DNAs were involved in major chromosomal rearrangements in both cases. Interestingly, recombination of the HBV Dna insert during the nude mouse passage had completely abolished HBV-specific transcription in one case, indicating that expression of integrated HBV genes, while presumably involved in early transformation, is dispensable at later stages of tumor progression. The sequential transformation observed in this experimental system suggests that expression of the X gene by integrated viral DNA and subsequent hepatocyte genome mutations might both contribute to HBV-associated liver carcinogenesis.

The HBV-producing cell line HepG2-4A5: a new in vitro system for studying the regulation of HBV replication and for screening anti-hepatitis B virus drugs.

All tissue culture systems for propagating HBV employed so far make use of tandemly arranged HBV genomes usually under the control of strong foreign promoters. Thus these systems are helpful for virus production but are of limited value in the investigation of the regulation of HBV replication or of the extent to which the expression of viral genes might be influenced by cellular signal transduction pathways. To overcome this barrier we established an HBV-producing cell line (HepG2-4A5) by stably transfecting HepG2 cells with a replication-competent, terminally redundant HBV plasmid (pSPT1.2 xHBV) that contains each of the four major HBV-ORFs only once and exclusively under the control of their own regulatory elements. HepG2-4A5 cells contain a single, nonrearranged, chromosomally integrated, replication-competent HBV genome. In the cytoplasm of HepG2-4A5 cells, all typical viral mRNAs were detectable, but no other viral transcripts were found. Furthermore, all viral gene products are synthesized in a balanced ratio, as close as possible to that found in an in vivo infection. Dane-like particles released from HepG2-4A5 cells were indistinguishable from virions synthesized in vivo, by all physical (electron microscopy, buoyant density) and biochemical (endogenous polymerase reaction, immunogenic behaviour) criteria. Because of the autologous genome organization in this system, the HepG2-4A5 cell line allows studies on the function of the HBV gene products with respect to their involvement in regulating HBV replication under conditions imitating as closely as possible the situation in vivo. Furthermore, this cell line might be a helpful tool in screening antiviral drugs and in studying their effect on regulating HBV replication.

Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses.

Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB.

Characterization of essential domains for the functionality of the MHBst transcriptional activator and identification of a minimal MHBst activator.

Integrated hepatitis B virus DNA derived from hepatocellular carcinomas can express, in one third of the cases investigated so far, a transcriptional activator encoded from 3' terminal truncated surface (preS/S) genes resulting in a C-terminally truncated middle surface protein (MHBst). Since MHBst, in contrast to the secreted MHBs, is retained in the secretory pathway at the ER, the question as to whether the retention generates the transcriptional activator function was investigated. Through fusion of MHBs to the ER-retention signal KDEL, it was shown that the intracellular retention does not generate the transcriptional activator function. Tryptic digestions of microsomal vesicles revealed that the amino terminal domain of MHBst directs into the cytoplasmic compartment, whereas in MHBs this domain directs into the lumen of the ER. This structural difference appears to be why transcriptional activator function arises. Through deletion analysis it was shown that non-membrane-associated MHBst proteins are also functional activators. Nonmembrane associated MHBst proteins represent a second class of MHBst proteins. These MHBst-proteins are homogenously distributed all over the cell and show no difference in functionality as compared to the membrane-associated MHBst proteins. MHBst53 (truncated at aa53) was shown to be a minimal activator of this class. Both classes of MHBst proteins were found to form dimers; an which is involved in mediating the dimerization. The integrity of this domain was also revealed to be a prerequisite for the functionality of the activator, suggesting a linkage between dimerization and functionality.

Round table discussion 'gene therapy and society'--summary and conclusions.

An international group consisting of scientists, physicians, and one lawyer reported on the problems of gene therapy with regard to society in their respective countries and discussed ethical, legal and scientific aspects of gene therapy. Ethical questions and fear in society were addressed by Dr Odenbach, former Secretary General of the German Medical Association, member of the German Parliamentary Enquiry Commission, 'Prospects and Risks of Gene Technology' and present adviser of the Ethical Committee of the World Medical Association, and by PH Hofschneider, Professor of Virology at the Max-Planck-Institut for Biochemistry, Munich, with a long-time strong interest in medical ethics; legal aspects were addressed by H Hausheer, Professor of Civil Law with many years experience in constitutional law and jurisdiction in Switzerland. Medical and scientific aspects were discussed by R Strohman, Emeritus Professor of Molecular Biology, Berkeley, USA, F Takaku, President of the International Medical Center of Japan and Chairman of the Central Evaluation Committee of the Japanese government for gene therapy, Tokyo and J Goldman, Professor of Leukemia Biology at Hammersmith Hospital, London, and known for this pioneering work in bone marrow transplantation. The discussion was moderated by R Hehlmann, Professor of Medicine at the University of Heidelberg and member of the World Committee of the International Association.

Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides.

The inhibitory effect of antisense phosphorothioate oligodeoxynucleotides (S-ODN) on hepatitis C viral gene expression and analyzed in an in vitro test system and in cell culture. S-ODN were directed against different stem loop structures in the 5'noncoding region (NCR) of the hepatitis C virus, (HCV) RNA and against a nucleotide stretch, including the start codon of the polyprotein precursor. The inhibitory effect of these S-ODN was quantified employing a viral RNA consisting of the first 407 nucleotides of a HCV type 1b genome fused to the coding sequence of the firefly luciferase gene. For in vitro assays this RNA was generated by in vitro transcription and used as a template in a rabbit reticulocyte lysate in vitro translation system. The production of active luciferase in the absence or presence of S-ODN was monitored using an enzymatic assay. The best results were obtained with S-ODN 4 directed against nucleotides 326 to 348, comprising the start AUG of the polyprotein coding sequence. With this oligonucleotide, a specific and dose-dependent effect was observed with a maximal inhibition of 96 +/- 1% at a S-ODN concentration of 4.14 mumol/L. For cell culture experiments, the hepatoblastoma cell line HepG2 was transfected with a plasmid expressing the HCV-luciferase fusion RNA. In this assay system S-ODN 2, complementary to nucleotides 264 to 282 of the HCV RNA, and S-ODN 4 were most efficient and reduced the viral translation by 96 +/- 0.4% and 94 +/- 0.7%, respectively, at a concentration of 0.3 mumol/L. The inhibition was specific (1) because the expression of the HCV-luciferase fusion RNA was not significantly impaired by the control S-ODN and (2) because the expression of an unrelated messenger RNA was not or only slightly downregulated. These data suggest that HCV gene expression can be inhibited effectively by antisense S-ODN. Therefore, this approach represents a promising perspective for the treatment of hepatitis C.

Transactivating function and expression of the x gene of hepatitis B virus.

BACKGROUND/AIMS: The x gene of hepatitis B virus encodes a transactivating factor of 154 amino acids, termed HBx, which stimulates transcription of multiple viral and cellular genes. The transactivating function is probably associated with a tumorigenic potential of HBx, since x gene sequences, encoding functional HBx, have been repeatedly found integrated into the genome of liver carcinoma cells. METHODS: To identify the transactivating domain of HBx, we constructed x gene plasmids encoding full length HBx or HBx fragments. We determined their transactivating function after cotransfection of cells, along with a plasmid that contains a reporter gene driven by the SV40 early promoter/enhancer region. RESULTS: Our results demonstrate that a 95-amino acid fragment of HBx, encompassing amino acids 49 to 143, contains all the elements that are required for the transactivating function. Within this fragment a sequence element, encompassing amino acids 107 to 130, which contains a relatively high number of amino acids with charged side chains, appears to be crucial for the stimulation of gene expression. The influence of deletion mutations on x mRNA steady-state levels and HBx stability was examined. In essence, stable RNA and protein were produced if at least codons 1-82 or 70-154 were present in the deletion plasmids. CONCLUSION: This finding strongly suggests that the deletion of functional domains between codons 49 and 143, but not an instability of RNA and/or protein, was critical for the loss of transactivation.

Identification of interleukin-1 and platelet-derived growth factor-B as major mitogens for the spindle cells of Kaposi's sarcoma: a combined in vitro and in vivo analysis.

By means of a combined in vitro and in vivo analysis we provide evidence that IL-1 beta and PDGF-B, but not OSM (oncostatin M) or IL-6, are major mitogens for the spindle cells of Kaposi's sarcoma (KS) in vivo. PDGF-B and IL-1 beta stimulated proliferation of cultivated KS spindle cells in vitro. Analysis of gene expression in vivo revealed that both factors as well as the PDGF beta-receptor are present in KS lesions. By contrast, IL-6 had no effect and OSM inhibited proliferation of cultivated KS spindle cells. Again, the effect of these factors on cultivated KS spindle cells in vitro was reflected by the gene expression observed in KS lesions in vivo. Neither the expression of IL-6 receptor nor of OSM could be detected in KS lesions by in situ hybridization. Moreover, in situ hybridization revealed an identical pattern of gene expression in cultivated KS spindle cells and KS spindle cells in vivo with respect to the above-mentioned cytokines [PDGF-B, IL-1 beta, IL-1 alpha, IL-6, OSM] and their receptors [PDGF beta-receptor, gp130, IL-6 receptor, leukemia inhibitory factor (LIF) receptor]. This further supported the suitability of cultivated KS spindle cells as an in vitro model in order to determine which cytokines may activate proliferation of KS spindle cells in vivo.

Enzymatic properties of overexpressed HBV-mevalonate kinase fusion proteins and mevalonate kinase proteins in the human hepatoma cell line PLC/PRF/5.

We have previously reported a case of integration of HBV sequences in the 5'-noncoding region of the gene for mevalonate kinase (Mk) (EC.2.7.1.36) in the human hepatoma cell line PLC/PRF/5, resulting in overexpression of viral-cellular fusion transcripts and enhanced intracellular levels of the enzyme. Here, we present an evaluation of the functionalities of Mk and HBV/Mk fusion proteins derived from viral-cellular fusion- and Mk-transcripts, some of which lack 156 bp in the Mk coding region as a result of a differential splicing process. cDNA clones with a full-length Mk-ORF produce proteins which can metabolize mevalonate to its monophosphorylated form. Our results suggest that the enhanced and inappropriate expression of Mk may lead to increased metabolism of mevalonate and phosphorylation of hitherto unknown cellular proteins. This consequence of HBV-DNA insertion could thus be related to the activation of proteins that may be relevant in oncogenesis.