Biomedical subjects

H Will

Publications and source records attributed to H Will.

At least 19 recordsLinked to original sources

PubMed2004

Spread of hepatitis B viruses in vitro requires extracellular progeny and may be codetermined by polarized egress.

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.

Animals↗

PubMed2003

A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wild-type virus.

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i). their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii). the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii). the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV.

Animals↗

PubMed2003

Body language: the function of PML nuclear bodies in apoptosis regulation.

Promyelocytic leukaemia (PML) nuclear bodies (NBs) are macromolecular nuclear domains present in virtually every mammalian cell. PML nuclear bodies (PML-NBs) were functionally linked to various fundamental cellular processes, including transcriptional control, tumour suppression and apoptosis regulation. Supporting the important function of PML and its associated NBs in apoptosis regulation, several apoptotic regulators localise to PML-NBs, and cells from PML-deficient mice show severe apoptotic defects, including induction of genotoxic stress and death receptor CD95 (Fas/APO-1) activation. Based on the current literature, we hypothesise that PML-NBs regulate apoptosis through different molecular mechanisms, on the one hand by acting as macromolecular scaffolds for recruitment and post-translational modification of the apoptotic key regulator p53, and on the other by regulating the subcellular bioavailability and quality of some apoptotic signal transducers.

Animals↗

PubMed2001

Identification and analysis of a new hepadnavirus in white storks.

We identified, cloned, and functionally characterized a new avian hepadnavirus infecting storks (STHBV). STHBV has the largest DNA genome of all avian hepadnaviruses and, based on sequence and phylogenetic analysis, is most closely related to, but distinct from, heron hepatitis B virus (HHBV). Unique for STHBV among the other avian hepadnaviruses is a potential HNF1 binding site in the preS promoter. In common only with HHBV, STHBV has a myristylation signal on the S and not the preS protein, two C terminally located glycosylation sites on the precore/core proteins and lacks the phosphorylation site essential for the transcriptional transactivation activity of duck-HBV preS protein. The cloned STHBV genomes were competent in gene expression, replication, and viral particle secretion. STHBV infected primary duck hepatocytes very inefficiently suggesting a restricted host range, similar to other hepadnaviruses. This discovery of stork infections unravels novel evolutionary aspects of hepadnaviruses and provides new opportunities for hepadnavirus research.

Amino Acid Sequence↗

PubMed2001

Structural and functional heterogeneity of naturally occurring hepatitis B virus variants.

Most organisms have developed sophisticated machineries to preserve their genomic integrity. On the contrary hepatitis B virus (HBV), like a lot of other viruses can undergo rapid and drastic sequence changes, especially if the virus has to cope with natural or therapy induced antiviral mechanisms in the host. Here, we try to summarize possible implications for the molecular pathogenesis of HBV based on the extensive research on the genetic variants of HBV.

Genes, Viral↗

PubMed2001

Generation of stable mRNA fragments and translation of N-truncated proteins induced by antisense oligodeoxynucleotides.

Binding of phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) to target mRNAs is commonly thought to mediate RNA degradation or block of translation. Here we demonstrate cleavage of target mRNAs within the AS ODN binding region with subsequent degradation of the 5' but not the 3' cleavage product. Some, if not all, 3' mRNA fragments lacked a 5' cap structure, whereas their poly(A) tail length remained unchanged. Furthermore, they were efficiently translated into N-terminally truncated proteins as demonstrated in three settings: production of shortened hepadnaviral surface proteins, alteration of the subcellular localization of a fluorescent protein, and shift of the transcription factor C/EBPalpha isoform expression levels. Thus, AS treatment may result in the synthesis of N-truncated proteins with biologically relevant effects.

Animals↗

PubMed2001

Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.

Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation.

Adult↗

PubMed2001

A dominant hepatitis B virus population defective in virus secretion because of several S-gene mutations from a patient with fulminant hepatitis.

There is increasing evidence that certain pathogenic hepatitis B virus (HBV) variants may play a role in the pathogenesis of fulminant hepatitis (FHB). Recently, we isolated from a patient with fulminant recurrent hepatitis B after liver transplantation variants with enhanced replication competence and a possible defect in viral particle secretion. Both viral features may have contributed to the severity of the disease. The aim of this study was to prove the secretion defect of these variants, to analyze the consequences, and to identify the responsible viral mutations. The variant genomes and appropriate wild-type/variant hybrid genomes were functionally characterized after transfection in human hepatoma cells. Two cloned genomes and the polymerase chain reaction (PCR)-amplified mixture of full-length genomes showed a block in viral particle secretion. This was caused by a combination of amino acid changes in the S-protein including the mutation G145R frequently emerging after hyperimmunoglobulin treatment. The mutations induced retention of the surface proteins in an endoplasmic reticulum (ER)-like compartment, but no intracellular accumulation. These data provide evidence for the in vivo existence of a dominant HBV population with a severe defect in viral particle secretion caused by mutations in the S-gene. This viral phenotype in combination with the enhanced replication competence may have contributed to the fulminant clinical course of the infection.

Amino Acid Substitution↗

PubMed2001

Woodchuck hepatocytes remain permissive for hepadnavirus infection and mouse liver repopulation after cryopreservation.

Isolated hepatocytes represent a relevant model of the liver and are highly required both for research and therapeutic applications. However, sources of primary liver cells from human beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be used for woodchuck hepatitis B virus (WHV) infection studies, and whether they could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of University of Wisconsin (UW) solution as a main component of the freezing medium, stepwise reduction of dimethylsulfoxide (DMSO) to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was still high (70% to 80%), and 50% to 60% of thawed cells attached to the plates. The appearance of covalently closed circular (ccc)DNA and of WHV-replicative forms a few days after in vitro infection demonstrated that thawed woodchuck hepatocytes were still susceptible to viral infection, thus proving maintenance of a very high hepatocyte-specific differentiation status. Furthermore, transplantation of woodchuck hepatocytes into the liver of urokinase-type plasminogen activator (uPA)/recombination activation gene-2 (RAG-2) mice, a model of liver regeneration, demonstrated that cryopreserved cells retained the ability to divide and to extensively repopulate a xenogenic liver. Notably, in vivo susceptibility to infection with WHV and proliferative capacity of frozen/thawed woodchuck hepatocytes in recipient mice were identical to those observed by transplanting fresh hepatocytes.

Animals↗

PubMed2001

Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame.

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

Amino Acid Sequence↗

PubMed2000

Lack of a role of the interferon-stimulated response element-like region in interferon alpha -induced suppression of Hepatitis B virus in vitro.

The antiviral effect of interferon-alpha (IFNalpha) on hepatitis B virus (HBV) is well documented in vitro and in vivo, but the mechanisms involved are elusive. Recently, an interferon-stimulated response like element (ISRE) competent for binding of interferon-stimulated gene factor-3gamma (p48) has been identified in the HBV enhancer I region. Mutation of this element was shown to abrogate IFNalpha-mediated reduction of HBV X-gene promoter-driven reporter gene expression. This suggested a role of the ISRE and of p48 in IFNalpha-induced antiviral activity against productive HBV infection. Here, we analyzed the antiviral effect of both IFNalpha and enhanced p48 expression on complete HBV genomes containing the wild-type or mutated ISRE. In human hepatoma cells transfected with both genomes, viral RNA and replicative intermediates were reduced by IFNalpha treatment to a similar degree. Enhanced p48 expression increased IFNalpha-induced suppression of HBV RNA significantly from 75 +/- 22.5% to 46 +/- 9.8%, but this was independent of the integrity of the ISRE-like region. These data imply that p48 neither mediates the antiviral activity of IFNalpha against HBV nor down-regulates enhancer I activity by binding directly to the HBV ISRE-like region, but rather argue for an indirect role of p48.

Antiviral Agents↗

PubMed2000

Regulation of p53 activity in nuclear bodies by a specific PML isoform.

Covalent modification of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a specific PML isoform (PML3) or by coexpression of SUMO-1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C-terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter-specific manner and affects cell survival. Our results indicate the existence of a cross-talk between PML- and p53-dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions.

Biological Transport, Active↗

PubMed2000

Enhanced replication contributes to enrichment of hepatitis B virus with a deletion in the core gene.

Accumulation in immunosuppressed patients of hepatitis B virus (HBV) with a deletion in the C gene is associated with severe liver disease. The aim of this study was to determine the phenotype of such genomes in vitro. Four C gene fragments with different types of deletions were inserted in the context of a wild-type genome and tested by transfection into HuH7 cells. The deletions did not influence mRNA and surface protein levels. Truncated C gene translation products were expressed only from variants with in-frame deletions, whereas full-length polymerase was expressed from all variants at a similar or higher level than in wild-type virus. None of the variants was competent for autonomous replication; however, they produced 2- to 4.5-fold more progeny DNA than wild-type HBV when sufficiently complemented with wild-type core protein. Similarly, when variant and wild-type DNA were cotransfected in different ratios, the variants produced 2- to 5-fold more progeny DNA relative to the wild-type; this enrichment required the expression of the viral polymerase in cis. The mechanism of enrichment depended on the percentage of variant in the transfected DNA mixture. When the transfected DNA contained a small percentage of variant, enhanced replication of the variant accompanied by no or little suppression of wild-type replication was seen. Accordingly, overall production of progeny virus was slightly increased. At a high percentage of variant DNA, replication of both variant and wild-type decreased, probably due to a shortage of wild-type core protein. In conclusion, emergence of C gene deletion variants in vivo may be due to enhanced replication mediated at the level of encapsidation or reverse transcription. If the variants constitute a small part of the ccc DNA, they can be fully trans-complemented by wild-type virus which may increase the overall virus production.

Cells, Cultured↗

PubMed2000

Genotype-specific synthesis and secretion of spliced hepatitis B virus genomes in hepatoma cells.

Hepatitis B virus-infected patients frequently have viral particles with DNA derived from differently spliced RNA. Which factors influence the synthesis of these splice variants is unclear. We analysed the type of splice variants produced from different genotypes and determined whether they are secreted as efficiently as wild-type virus. We demonstrate production of a single splice variant from genotypes D, C, and E as dominant species in two hepatoma cell lines. The type of minor splice variants synthesised varied between genotypes but was identical in both hepatoma cell lines. A novel splice variant with a deletion in the core gene was identified for genotype D. Viral DNA from intracellular compared with extracellular viral particles was spliced approximately five times more often than wild-type-sized genomes. A variable amount of the major splice variant was also identified in sera from patients infected with genotypes A, D, and C. These data indicate genotype A-, C-, D-, and E- as well as hepatoma cell line-independent synthesis of a dominant single splice variant and argue for a biological function of the corresponding splice sites. This study clearly demonstrates the intracellular accumulation of viral particles containing spliced genomes and offers a tool for the investigation of underlying mechanisms.

Alternative Splicing↗

PubMed2000

The autoimmune regulator protein has transcriptional transactivating properties and interacts with the common coactivator CREB-binding protein.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, caused by mutations in the autoimmune regulator (AIRE) gene, is an autosomal recessive autoimmune disease characterized by the breakdown of tolerance to organ-specific antigens. The 545 amino acid protein encoded by AIRE contains several structural motifs suggestive of a transcriptional regulator and bears similarity to cellular proteins involved in transcriptional control. We show here that AIRE fused to a heterologous DNA binding domain activates transcription from a reporter promoter, and the activation seen requires the full-length protein or more than one activation domain. At the structural level AIRE forms homodimers through the NH(2)-terminal domain, and molecular modeling for this domain suggests a four-helix bundle structure. In agreement, we show that the common transcriptional coactivator CREB-binding protein (CBP) interacts with AIRE in vitro and in yeast nuclei through the CH1 and CH3 conserved domains. We suggest that the transcriptional transactivation properties of AIRE together with its interaction with CBP might be important in its function as disease-causing mutations almost totally abolish the activation effect.

Amino Acid Sequence↗

PubMed2000

The tetracycline-responsive promoter contains functional interferon-inducible response elements.

Tetracycline (tet)-responsive expression vectors allow controlled inducible expression of proteins in mammalian cells. This system is widely used for experimental research both in vivo and in vitro. In our attempts to use this system to study the antiviral effect of IFNalpha on hepatitis B virus, we discovered an unexpected feature of the tet-responsive promoter (tet promoter) of the currently available expression vectors. IFNalphawas found to stimulate tet promoter activity after transient transfection in a dose- and cell type-dependent manner. By sequence inspection, an IFNalpha-stimulated response element (ISRE)-like sequence was identified in the linker regions located between the heptameric tet operator sequences. Gel shift assays revealed binding of IFN-stimulated gene factors to these sequences, indicating that they mediate the IFNalpha-mediated promoter stimulation. These data demonstrate an unexpected feature of the tet-responsive expression system which needs to be taken into account when using this system for analysis of cytokine functions in vitro and in vivo. The data also imply that the tet promoter-based expression system can be rendered non-responsive to IFNalpha by mutagenesis of the ISREs and this may be essential when considering gene therapy in vivo.

Anti-Bacterial Agents↗

PubMed2000

Serological pattern "anti-HBc alone": report on a workshop.

In areas with low hepatitis B virus (HBV) endemicity such as most parts of Europe and the United States "anti-HBc alone" is found in 10-20% of all individuals with HBV markers, i.e., 1-4% of the population. In about 10% of these individuals HBV DNA is detected by PCR, the proportions varying greatly depending on the population studied, being highest in individuals coinfected with hepatitis C virus (HCV) (above 35%) and HIV (above 85%). A small proportion of individuals with "anti-HBc alone" are in the window phase of an HBV infection or in a stage of late HBV immunity. For the large proportion of these individuals this is not the case and they are thought to have an unresolved HBV-infection or a chronic infection in a late or "low grade" productive state. Currently, limited studies have been performed concerning the clinical aspects of individuals with "anti-HBc alone" and suspected chronic HBV infection. The majority of these individuals seem to be healthy. Some chronic carriers with "anti-HBc alone," however, do present signs of chronic hepatitis. Individuals with "anti-HBc alone" are potentially infectious. This is exemplified by a few case reports of HBV transmission to sexual contacts, perinatal transmission between mother and newborns and in blood recipients. Recommendations are given in relation to both the diagnostic and therapeutic procedures in the individuals with "anti-HBc alone" and in the blood banking and transplantation services.

Blood Banks↗

PubMed2000

Personality and object relations in patients with affective disorders: idiographic research by means of the repertory grid technique.

BACKGROUND: This paper presents an idiographic approach to evaluate the self concept and the self-object-relationship of patients suffering from affective disorders. METHODS: Significant dimensions of the personality and the object relations of 127 depressive patients and 34 orthopaedic patients were investigated with the repertory grid-technique. The self concept and the object relations were compared by means of nomothetically used idiographic results after recovery from manifest depression. RESULTS: 'Low self esteem' was frequently found in patients with a long lasting course of illness and the ICD-10-diagnoses of 'bipolar affective disorder' and 'dysthymia'. The object relations of the depressive sample were characterised by the dimension 'symbiotic near'; 'ambivalent' and 'indifferent' partnership relationships were found much more frequently in the controls. CONCLUSIONS: The idiographic results help to differentiate the spectrum of affective disorders. They underline the importance of the interpersonal dimension of depression and may be used as a basis of a therapeutic appraisal. LIMITATIONS: The repertory grid-technique may not be used as a diagnostic instrument. However, the combination of idiographic results with further clinical informations enables the multidimensional assessment of the self concept and psychosocial coping mechanisms.

Adaptation, Psychological↗